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SUMMARYGene editing theBCL11Aerythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of theBCL11A+58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of theBCL11A+58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.

Antibody heavy chain (HC) and light chain (LC) variable region exons are assembled by V(D)J recombination. V(D)J junctional regions encode complementarity-determining-region 3 (CDR3), an antigen-contact region immensely diversified through non-templated nucleotide additions (“N-regions”) by terminal deoxynucleotidyl transferase (TdT). HIV-1 vaccine strategies seek to elicit human HIV-1 broadly neutralizing antibodies (bnAbs), such as the potent CD4-binding site VRC01-class bnAbs. Mice with primary B cells that express receptors (BCRs) representing bnAb precursors are used as vaccination models. VRC01-class bnAbs uniformly use human HC VH1-2 and commonly use human LCs Vκ3-20 or Vκ1-33 associated with an exceptionally short 5-amino-acid (5-aa) CDR3. Prior VRC01-class models had non-physiological precursor levels and/or limited precursor diversity. Here, we describe VRC01-class rearranging mice that generate more physiological primary VRC01-class BCR repertoires via rearrangement of VH1-2, as well as Vκ1-33 and/or Vκ3-20 in association with diverse CDR3s. Human-like TdT expression in mouse precursor B cells increased LC CDR3 length and diversity and also promoted generation of shorter LC CDR3s via N-region suppression of dominant microhomology-mediated Vκ-to-Jκ joins. Priming immunization with eOD-GT8 60mer, which strongly engages VRC01 precursors, induced robust VRC01-class germinal center (GC) B cell responses. Vκ3-20-based responses were enhanced by N-region addition, which generates Vκ3-20-to-Jκ junctional sequence combinations that encode VRC01-class 5-aa CDR3s with a critical E residue. VRC01-class-rearranging models should facilitate further evaluation of VRC01-class prime and boost immunogens. These new VRC01-class mouse models establish a prototype for generation of vaccine-testing mouse models for other HIV-1 bnAb lineages that employ different HC or LC Vs.Significance StatementMouse models that express human precursors of HIV-1 broadly neutralizing antibodies (bnAbs) are useful for evaluating vaccination strategies for eliciting such bnAbs in humans. Prior models were handicapped by non-physiological frequency and/or diversity of B lymphocytes that express the bnAb precursors. We describe a new class of mouse models in which the mice express humanized bnAb precursors at a more physiologically relevant level through developmental rearrangement of both antibody heavy and light chain gene segments that encode the precursors. The model also incorporated a human enzyme that diversifies the rearranging gene segments and promotes generation of certain variable region sequences needed for the response. This new class of mouse models should facilitate preclinical evaluation of candidate human HIV-1 vaccination strategies.

In this work we provide preclinical data to support initiation of a first-in-human trial for sickle cell disease (SCD) using an approach that relies on reversal of the developmental fetal-to-adult hemoglobin switch. Erythroid-specific knockdown of BCL11A via a lentiviral-encoded microRNA-adapted short hairpin RNA (shRNAmiR) leads to reactivation of the gamma-globin gene while simultaneously reducing expression of the pathogenic adult sickle β-globin. We generated a refined lentiviral vector (LVV) BCH-BB694 that was developed to overcome poor vector titers observed in the manufacturing scale-up of the original research-grade LVV. Healthy or sickle cell donor CD34+ cells transduced with Good Manufacturing Practices (GMP)-grade BCH-BB694 LVV achieved high vector copy numbers (VCNs) >5 and gene marking of >80%, resulting in a 3- to 5-fold induction of fetal hemoglobin (HbF) compared with mock-transduced cells without affecting growth, differentiation, and engraftment of gene-modified cells in vitro or in vivo. In vitro immortalization assays, which are designed to measure vector-mediated genotoxicity, showed no increased immortalization compared with mock-transduced cells. Together these data demonstrate that BCH-BB694 LVV is non-toxic and efficacious in preclinical studies, and can be generated at a clinically relevant scale in a GMP setting at high titer to support clinical testing for the treatment of SCD.

Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and β-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB −28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification. A single therapeutic base edit of the BCL11A enhancer in human HSPCs can ameliorate cellular defects associated with sickle cell disease and β-thalassemia in vitro and efficiently induce fetal hemoglobin expression upon engraftment in mice in vivo.

A promising treatment for β-hemoglobinopathies is the de-repression of γ-globin expression leading to increased fetal hemoglobin (HbF) by targeting BCL11A. Here, we aim to improve a lentivirus vector (LV) containing a single BCL11A shmiR (SS) to further increase γ-globin induction. We engineered a novel LV to express two shmiRs simultaneously targeting BCL11A and the γ-globin repressor ZNF410. Erythroid cells derived from human HSCs transduced with the double shmiR (DS) showed up to a 70% reduction of both BCL11A and ZNF410 proteins. There was a consistent and significant additional 10% increase in HbF compared to targeting BCL11A alone in erythroid cells. Erythrocytes differentiated from SCD HSCs transduced with the DS demonstrated significantly reduced in vitro sickling phenotype compared to the SS. Erythrocytes differentiated from transduced HSCs from β-thalassemia major patients demonstrated improved globin chain balance by increased γ-globin with reduced microcytosis. Reconstitution of DS-transduced cells from Berkeley SCD mice was associated with a statistically larger reduction in peripheral blood hemolysis markers compared with the SS vector. Overall, these results indicate that the DS LV targeting BCL11A and ZNF410 can enhance HbF induction for treating β-hemoglobinopathies and could be used as a model to simultaneously and efficiently target multiple gene products.

Clinical outcomes from granulocyte transfusion (GTX) are disadvantaged by the short shelf life and compromised function of donor neutrophils. Spontaneous neutrophil death is heterogeneous and mediated by multiple pathways. Leveraging mechanistic knowledge and pharmacological screening, we identified a combined treatment, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which altered neutrophil fate by simultaneously targeting multiple cell death pathways. CLON-G prolonged human and mouse neutrophil half-life in vitro from less than 1 day to greater than 5 days. CLON-G-treated aged neutrophils had equivalent morphology and function to fresh neutrophils, with no impairment to critical effector functions including phagocytosis, bacterial killing, chemotaxis, and reactive oxygen species production. Transfusion with stored CLON-G-treated 3-day-old neutrophils enhanced host defenses, alleviated infection-induced tissue damage, and prolonged survival as effectively as transfusion with fresh neutrophils in a clinically relevant murine GTX model of neutropenia-related bacterial pneumonia and systemic candidiasis. Last, CLON-G treatment prolonged the shelf life and preserved the function of apheresis-collected human GTX products both ex vivo and in vivo in immunodeficient mice. Thus, CLON-G treatment represents an effective and applicable clinical procedure for the storage and application of neutrophils in transfusion medicine, providing a therapeutic strategy for improving GTX efficacy.

While cancer is an age-related disease, most studies focus on genetically engineered younger mouse models. Here, we uncover how cancer develops as a consequence of the naturally aged immune system in mice. B-cell lymphoma frequently occurs in aged mice and is associated with increased cell size, splenomegaly, and a novel clonal B-cell population. Age-emergent B cells clonally expand outside of germinal centers driven by somatic mutations, activated c-Myc and hypermethylated promoters, and both genetically and epigenetically recapitulate human follicular and diffuse-large B-cell lymphomas. Mechanistically, mouse cancerous B cells originate from age-associated B cells, which are atypical memory B cells. Age-associated B cells secrete a spectrum of proinflammatory cytokines and activate paracrinally the expression of c-Myc in surrounding B cells. Although clonal B cells are a product of an aging microenvironment, they evolve being self-sufficient and support malignancy when transferred into young mice. Inhibition of mTOR and c-Myc attenuates premalignant changes in B cells during aging and emerges as a therapeutic strategy to delay the onset of age-related lymphoma. Together, we uncover how aging generates cancerous B cells, characterize a model that captures the origin of spontaneous cancer during aging and identify interventions that may postpone age-associated lymphoma.

Emerging cellular therapies require the collection of peripheral blood hematopoietic stem cells (HSC) by apheresis for in vitro manipulation to accomplish gene addition or gene editing. These therapies require relatively large numbers of HSCs within a short time frame to generate an efficacious therapeutic product. This review focuses on the principal factors that affect collection outcomes, especially relevant to gene therapy for sickle cell disease.

Sickle cell disease is characterized by hemolytic anemia, pain, and progressive organ damage. A high level of erythrocyte fetal hemoglobin (HbF) comprising α- and γ-globins may ameliorate these manifestations by mitigating sickle hemoglobin polymerization and erythrocyte sickling.We enrolled patients with sickle cell disease in a single-center, open-label pilot study. The investigational therapy involved infusion of autologous CD34+ cells transduced with the BCH-BB694 lentiviral vector, which encodes a short hairpin RNA (shRNA) targetingAs of October 2020, six patients had been followed for at least 6 months after receiving BCH-BB694 gene therapy; median follow-up was 18 months (range, 7 to 29). All patients had engraftment, and adverse events were consistent with effects of the preparative chemotherapy. All the patients who could be fully evaluated achieved robust and stable HbF induction (percentage HbF/(F+S) at most recent follow-up, 20.4 to 41.3%), with HbF broadly distributed in red cells (F-cells 58.9 to 93.6% of untransfused red cells) and HbF per F-cell of 9.0 to 18.6 pg per cell. Clinical manifestations of sickle cell disease were reduced or absent during the follow-up period.This study validates BCL11A inhibition as an effective target for HbF induction and provides preliminary evidence that shmiR-based gene knockdown offers a favorable risk-benefit profile in sickle cell disease. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT03282656).

Protein phosphatase 2A (PP2A), a serine/threonine phosphatase, has been shown to control T cell function. We found that in vitro–activated B cells and B cells from various lupus-prone mice and patients with systemic lupus erythematosus display increased PP2A activity. To understand the contribution of PP2A to B cell function, we generated a Cd19(Cre)Ppp2r1a(fl/fl) (flox/flox) mouse which lacks functional PP2A only in B cells. Flox/flox mice displayed reduced spontaneous germinal center formation and decreased responses to T cell-dependent and T-independent antigens, while their B cells responded poorly in vitro to stimulation with an anti-CD40 antibody or CpG in the presence of IL-4. Transcriptome and metabolome studies revealed altered nicotinamide adenine dinucleotide (NAD) and purine/pyrimidine metabolism and increased expression of purine nucleoside phosphorylase in PP2A-deficient B cells. Our results demonstrate that PP2A is required for optimal B cell function and may contribute to increased B cell activity in systemic autoimmunity.

Summary Hematopoietic stem and progenitor cells (HSPCs) undergo self-renewal and differentiation to guarantee a constant supply of short-lived blood cells. Both intrinsic and extrinsic factors determine HSPC fate, but the underlying mechanisms remain elusive. Here, we report that Proteinase 3 (PR3), a serine protease mainly confined to granulocytes, is also expressed in HSPCs. PR3 deficiency intrinsically suppressed cleavage and activation of caspase-3, leading to expansion of the bone marrow (BM) HSPC population due to decreased apoptosis. PR3-deficient HSPCs outcompete the long-term reconstitution potential of wild-type counterparts. Collectively, our results establish PR3 as a physiological regulator of HSPC numbers. PR3 inhibition is a potential therapeutic target to accelerate and increase the efficiency of BM reconstitution during transplantation., Highlights • Proteinase 3 (PR3) is expressed in hematopoietic stem and progenitor cells (HSPCs) • Deficiency of PR3 leads to expansion of HSPCs in murine bone marrow • PR3 regulates spontaneous HSPC apoptosis by cleaving and activating caspase-3, In this article, Luo and colleagues show that Proteinase 3 (PR3), a member of neutrophil serine proteases, is expressed in HSPCs and regulates HSPC numbers in murine bone marrow. Their results suggest that PR3 deficiency does not affect HSPC proliferation but reduces the rate of spontaneous HSPC apoptosis by cleaving and activating caspase-3.

Novel therapies for sickle cell disease (SCD) based on genetically engineered autologous hematopoietic stem and progenitor cells (HSPCs) are critically dependent on a safe and effective strategy for cell procurement. We sought to assess the safety and efficacy of plerixafor when used in transfused patients with SCD for HSC mobilization. Six adult patients with SCD were recruited to receive a single dose of plerixafor, tested at lower than standard (180 µg/kg) and standard (240 µg/kg) doses, followed by CD34+ cell monitoring in peripheral blood and apheresis collection. The procedures were safe and well-tolerated. Mobilization was successful, with higher peripheral CD34+ cell counts in the standard vs the low-dose group. Among our 6 donors, we improved apheresis cell collection results by using a deep collection interface and starting apheresis within 4 hours after plerixafor administration. In the subjects who received a single standard dose of plerixafor and followed the optimized collection protocol, yields of up to 24.5 × 106 CD34+ cells/kg were achieved. Interestingly, the collected CD34+ cells were enriched in immunophenotypically defined long-term HSCs and early progenitors. Thus, we demonstrate that plerixafor can be employed safely in patients with SCD to obtain sufficient HSCs for potential use in gene therapy.

Hypomorphic RAG1 mutations allowing residual T- and B-cell development have been found in patients presenting with delayed-onset combined immune deficiency with granulomas and/or autoimmunity (CID-G/AI) and abnormalities of the peripheral T- and B-cell repertoire. To examine how hypomorphic Rag1 mutations affect the earliest stages of lymphocyte development, we used CRISPR/Cas9 to generate mouse models with mutations equivalent to those found in patients with CID-G/AI. Immunological characterization showed partial development of T and B lymphocytes, with persistence of naive cells and preserved serum immunoglobulin but impaired antibody responses and presence of autoantibodies, thereby recapitulating the phenotype seen in patients with CID-G/AI. By using high-throughput sequencing, we identified marked skewing of Igh V and Trb V gene usage in early progenitors, with a bias for productive Igh and Trb rearrangements after selection occurred and increased apoptosis of B-cell progenitors. Rearrangement at the Igk locus was impaired, and polyreactive immunoglobulin M antibodies were detected. This study provides novel insights into how hypomorphic Rag1 mutations alter the primary repertoire of T and B cells, setting the stage for immune dysregulation frequently seen in patients.

Targeting the BCL11A erythroid enhancer by gene editing is a promising approach to fetal hemoglobin induction for beta-hemoglobinopathies. HbF levels vary widely among individuals, suggesting potential heterogeneity in HbF responses after therapeutic intervention. We hypothesize that maximizing both gene edit frequency and HbF induction potential could promote consistently favorable clinical outcomes. Here we compared CRISPR-Cas9 endonuclease editing of the BCL11A +58 enhancer with alternative gene modification approaches, including +55 erythroid enhancer editing alone or in combination with the +58 enhancer, as well as editing targeting the HBG1/2 promoter -115 BCL11A binding site and transduction by an shRNA knocking down the BCL11A transcript in erythroid precursors. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in the most potent HbF induction (52.4%±6.3%) of tested approaches (BCL11A +58 editing alone, 29.1%±3.9%; BCL11A +55 editing alone, 34.8±5.1%; HBG1/2 promoter editing, 34.1% ±5.4%; shmiR-BCL11A, 32.2%±4.4%; mock, 7.6%±3.4%). Based on assays in bulk and single cell derived erythroid cultures and xenografted immunodeficient mice, we found that disruption of core half E-box/GATA motifs at both the +58 and +55 enhancers was associated with greatest HbF induction, whether by small indels, interstitial 3.1 kb deletion, or 3.1 kb inversion. Rare gene edited clones with alleles that only partially disrupted these motifs were associated with intermediate HbF induction phenotypes. Combined editing of BCL11A +58 and +55 enhancers was compatible with HSC self-renewal in primary and secondary xenotransplant, with intact lymphoid, myeloid and erythroid repopulation. We conducted gene-edited cell product manufacturing process development and developed conditions using a MaxCyte electroporation instrument achieving mean 97.3±1.8% gene edits and 88.9%±6.4% viability 24 hours after electroporation in 3 engineering runs at clinical scale. We obtained similar results at small-scale with plerixafor-mobilized HSPCs from sickle cell disease (SCD) donors or G-CSF mobilized PBMCs from transfusion-dependent beta-thalassemia (TDT) donors, including 94.2%±4.4%, 99.5%±0.3% and 91.8%±6.3% of gene edits in engrafting cells from NBSGW 16 week mouse bone marrow of healthy, SCD and TDT donors respectively. Off-target analyses by pooled amplicon sequencing of 601 candidate off-target sites for the +58 and +55 targeting sgRNAs, nominated by a range of computational (CRISPRme) and experimental (GUIDE-seq and ONE-seq) methods, did not identify reference genome off-target edits at a sensitivity of 0.1% allele frequency. We evaluated +58/+55 enhancer combined targeting in nonhuman primates by performing ribonucleoprotein (RNP) electroporation in rhesus macaque mobilized peripheral blood CD34+ HSPCs with autologous re-infusion following busulfan myeloablation. We observed highly efficient gene edit frequency (85.2%, 88.8% and 84.9%) and durable HbF induction (26.4%, 57.5%, and 45.9% F-cells and 12.7%, 41.9%, and 28% gamma-globin) in the peripheral blood in 3 animals at most recent recorded time point post infusion (127, 78, and 54 weeks respectively). Single colony analyses and bulk ddPCR and unidirectional sequencing demonstrated that the long-term engrafting cells displayed a similar distribution of indels, 3.1 kb deletions, and 3.1 kb inversions as the input cell products. Erythroid stress due to hydroxyurea treatment, with or without phlebotomy, was associated with substantially augmented HbF responses (to 75.9%, 88.2%, and 57.8% F-cells and 47.9%, 68%, and 35.7% gamma-globin). No hematologic or other toxicities attributable to gene editing were observed. Together these results suggest that combined BCL11A +58 and +55 erythroid enhancer editing produces highly efficient on-target allelic disruption, erythroid-specific BCL11A downregulation, heightened HbF induction capacity compared to alternative approaches, preserved long-term multilineage engraftment potential by both human xenotransplant and rhesus autotransplant assays, and absence of evident genotoxicity, under clinically relevant SpCas9 RNP electroporation conditions. Disclosures No relevant conflicts of interest to declare.

Loss-of-function mutations in the signal transducer and activator of transcription 3 gene ( stat3 ) result in autosomal dominant hyper-IgE syndrome (AD-HIES), a condition in which patients have recurrent debilitating infections, including frequent pneumococcal and staphylococcal pneumonias. stat3 mutations cause defective adaptive T H 17 cellular responses, an immune mechanism believed to be critical for clearance of pneumococcal colonization and diminished antibody responses. Here we wished to evaluate the role of stat3 in the clearance of pneumococcal carriage and immunity using mice with a stat3 mutation recapitulating AD-HIES. We show here that naive AD-HIES mice have prolonged nasal carriage of pneumococcus compared to WT mice. Mutant and wild-type mice were then immunized with a pneumococcal whole-cell vaccine (WCV) that provides T H 17-mediated protection against pneumococcal colonization and antibody-mediated protection against pneumonia and sepsis. WCV-immunized AD-HIES mice made significantly less pneumococcus-specific interleukin-17A (IL-17A) and antibody than WT mice. The WCV-elicited protection against colonization was abrogated in AD-HIES mice, but immunization with WCV still protected AD-HIES mice against aspiration pneumonia/sepsis. Taken together, our results suggest that impaired clearance of nasopharyngeal carriage due to poor adaptive IL-17A responses may contribute to the increased rates of pneumococcal respiratory infection in AD-HIES patients.

Mooster et al. created a knock-in mouse harboring a mutation (S32I) in IκBα that has been identified in a patient with ectodermal dysplasia with immunodeficiency. The mice are characterized by defective architectural cell function; they lack lymph nodes, Peyer’s patches, splenic marginal zones, and follicular DCs and fail to develop germinal centers. These features have not been previously recognized in patients., Patients with ectodermal dysplasia with immunodeficiency (ED-ID) caused by mutations in the inhibitor of NF-κB α (IκBα) are susceptible to severe recurrent infections, despite normal T and B cell numbers and intact in vitro lymphocyte function. Moreover, the outcome of hematopoietic stem cell transplantation (HSCT) in these patients is poor despite good engraftment. Mice heterozygous for the IκBα S32I mutation found in patients exhibited typical features of ED-ID. Strikingly, the mice lacked lymph nodes, Peyer’s patches, splenic marginal zones, and follicular dendritic cells and failed to develop contact hypersensitivity (CHS) or form germinal centers (GCs), all features not previously recognized in patients and typical of defective noncanonical NF-κB signaling. Lymphotoxin β receptor (LTβR)–driven induction of chemokines and adhesion molecules mediated by both canonical and noncanonical NF-κB pathways was impaired, and levels of p100 were markedly diminished in the mutant. IκBα mutant→Rag2−/−, but not WT→IκBα mutant, bone marrow chimeras formed proper lymphoid organs and developed CHS and GCs. Defective architectural cell function explains the immunodeficiency and poor outcome of HSCT in patients with IκBα deficiency and suggests that correction of this niche is critical for reconstituting their immune function.

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Volpi et al. demonstrate that hypomorphic EXTL3 mutations cause abnormalities of heparan sulfate composition, affect signaling in response to growth factors and cytokines, and perturb thymopoiesis, resulting in a novel genetic disease associating skeletal dysplasia, T cell immunodeficiency, and neurodevelopmental delay., We studied three patients with severe skeletal dysplasia, T cell immunodeficiency, and developmental delay. Whole-exome sequencing revealed homozygous missense mutations affecting exostosin-like 3 (EXTL3), a glycosyltransferase involved in heparan sulfate (HS) biosynthesis. Patient-derived fibroblasts showed abnormal HS composition and altered fibroblast growth factor 2 signaling, which was rescued by overexpression of wild-type EXTL3 cDNA. Interleukin-2–mediated STAT5 phosphorylation in patients’ lymphocytes was markedly reduced. Interbreeding of the extl3-mutant zebrafish (box) with Tg(rag2:green fluorescent protein) transgenic zebrafish revealed defective thymopoiesis, which was rescued by injection of wild-type human EXTL3 RNA. Targeted differentiation of patient-derived induced pluripotent stem cells showed a reduced expansion of lymphohematopoietic progenitor cells and defects of thymic epithelial progenitor cell differentiation. These data identify EXTL3 mutations as a novel cause of severe immune deficiency with skeletal dysplasia and developmental delay and underline a crucial role of HS in thymopoiesis and skeletal and brain development.

Lrrc8a is a ubiquitously expressed gene that encodes a leucine-rich repeat (LRR)–containing protein detected at higher levels on the surface of thymocytes than on other immune cells. We generated Lrrc8a−/− mice to investigate the role of LRRC8A in lymphocyte development and function. Lrrc8a−/− mice had increased prenatal and postnatal mortality, growth retardation, and multiple tissue abnormalities. Lrrc8a−/− mice displayed a modest block in B cell development but intact intrinsic B cell function. In contrast, both Lrrc8a−/− mice and Lrrc8a−/−→Rag2−/− bone marrow chimeras exhibited a severe cell-intrinsic block in early thymic development, with decreased proliferation and increased apoptosis of thymocytes, and impaired peripheral T cell function. Thymic epithelial cells expressed an LRRC8A ligand that was critical for double-negative to double-positive thymocyte differentiation and survival in vitro. LRRC8A constitutively associated with the GRB2–GAB2 complex and lymphocyte-specific protein tyrosine kinase (LCK) in thymocytes. LRRC8A ligation activated AKT via the LCK–ZAP–70–GAB2–PI3K pathway, and AKT phosphorylation was markedly reduced in the thymus of Lrrc8a−/− mice. These findings reveal an essential role for LRRC8A in T cell development, survival, and function.

BCL11A regulates the fetal-adult hemoglobin switch by repressing expression at the gamma (γ)-globin locus (Sankaran et al., Science, 2008), and thus it represents an appealing therapeutic target for sickle cell disease (SCD). BCH-BB694 is a lentiviral vector (LVV) encoding a shRNA targeting BCL11A embedded in a microRNA scaffold (shmiR) allowing erythroid-specific knockdown to induce γ-globin expression and concomitantly and coordinately repress β-sickle globin expression (Brendel et al. JCI, 2016). In a pilot and feasibility gene therapy study we are evaluating the safety of infusion of BCH-BB694-transduced autologous CD34+ cells in patients with severe SCD. The study is an IND enabled and IRB approved open label, non-randomized, single center trial (NCT 03282656). We report here data from the full adult cohort which has completed enrollment with > 6 months of follow up in all patients. The adult cohort included three patients >/= 18 years old. Autologous CD34+ cells were collected by plerixafor mobilization and then transduced ex vivo with the BCH-BB694 shmiR lentiviral vector. Cell doses and vector copy number (VCN) are shown in the Table. After testing and release, gene modified cells were infused into subjects who had received busulfan conditioning. There were no Grade 3 or 4 AEs associated with mobilization, collection or infusion. All three adults (age 21-26 years old) demonstrated neutrophil engraftment on day +22 with adverse events consistent with busulfan conditioning. These patients are now 7, 9, and 17 months post infusion. One subject resumed red cell transfusions at 3 months due to pre-existing moyamoya using a pre-defined conservative trigger value of 40% sickle Hb in whole blood and will be detailed separately. There have been no adverse events related to the gene therapy product. VCN has been stable in bone marrow (BM) and peripheral blood (PB) in all cell lineages during the length of the study, with the latest time point studied at 15 months (BCL002) and ranged from 0.45-2.85 copies per cell in erythroid progenitor cells. BCL11A protein levels evaluated by immunoblot in subject BCL002 at 30 days (PB) and 6 months (BM) post-infusion showed highly effective and selective knockdown of BCL11A in erythroid progenitors with no reduction in BCL11A expression in B lymphoid cells. The number of HbF-containing cells (F cells) was assessed by flow cytometry and the kinetics of F cell production was remarkably similar in all subjects. The two untransfused subjects (BCL002 and BCL004) produced 70% F-cells in PB at 3 and 5 months, which has remained stable until the last point assayed (15 months and 7.5 months, respectively) (table). Calculated average HbF per F cell was >10pg in all subjects (table) and quantitative single cell HbF flow analysis showed the majority of F cells had >4pg F/cell, a level that is believed to prevent sickling under physiological oxygen saturation (Rakotoson et al., ASH 2017). In both untransfused subjects, total Hb remained stable with evidence of reduced hemolysis by reticulocyte count (slightly elevated) and LDH (normal in one subject, slightly elevated in the other). At the 3-month timepoint before re-starting transfusions, the subject with moyamoya (BCL003) had a pre-transfusion Hb of 11 g/dL with 76% of non-transfused cells containing on average 17pg F/F cell. For all subjects, we estimated the fraction of RBCs containing significant Hb sickle polymers and the amount of polymer in each sickled RBC at physiologic oxygen tension (where 50% of monomeric hemoglobin was oxygen saturated, or the P50) (Di Caprio et al. PNAS 2019, in press). The results for all 3 subjects in this adult cohort showed fewer RBCs with significant Hb polymer than two hydroxyurea-responsive treated comparators and significantly less Hb polymer per sickled RBC than a third highly responsive hydroxyurea-treated comparator. In conclusion, these data demonstrate successful and sustained engraftment in three adult patients treated with LVV-delivered shmiR technology targeting BCL11A. Early results suggest an acceptable safety profile, validation of BCL11A as effective target for HbF induction in humans with high numbers of F cells in circulation containing high levels of HbF per F cell, and mitigation of cellular pathology of SCD. Disclosures Achebe: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacosmos: Membership on an entity's Board of Directors or advisory committees; Fulcrum Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Bartolucci:Novartis: Membership on an entity's Board of Directors or advisory committees; AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; HEMANEXT: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Heeney:AstraZeneca: Research Funding; Micelle Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Research Funding; Ironwood / Cyclerion: Research Funding; Vertex / Crisper Therapeutics: Other: Data Safety Monitoring Board. Higgins:Sanofi: Consultancy, Research Funding. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Wood:Sanofi: Consultancy, Research Funding. Williams:Alerion Biosciences: Other: Co-founder; Novartis: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, Patents & Royalties: Potential for future royalty/milestone income, X-SCID., Research Funding; bluebird bio: Patents & Royalties: Licensed certain IP relevant to hemoglobinopathies to bluebird bio. Received payment in the past bluebird bio through a BCH institutional licensing agreement and there is a potential for future royalty/milestone income from this agreement., Research Funding.

Base editing with fusions of RNA-guided DNA-binding proteins and nucleotide deaminases represents a promising approach to permanently remedy genetic blood disorders without obligatory double strand breaks, however its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. Here we purified A3A(N57Q)-BE3 protein for RNP electroporation of human peripheral blood (PB) mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We found that sgRNAs targeting for cytidine base editing the core GATA1 binding motif of the BCL11A +58 erythroid enhancer resulted in efficient on-target base edits (81% allele frequency) with low indels. There was similar HbF induction in erythroid progeny as compared to Cas9:sgRNA RNP nuclease mediated modification of the same target sequence (36% median HbF in base edited cells with 81% allele modifications, 39% HbF in 3xNLS-SpCas9:sgRNA#1617 nuclease edited cells with 99% indels, and 5%HbF in unedited cells). A single therapeutic base edit of the BCL11A enhancer was sufficient to ameliorate the pathobiology of both sickle cell disease (SCD) and beta-thalassemia. Base editing of CD34+ HSPCs from plerixafor mobilized PB SCD patient donors resulted in potent HbF induction (24-31% HbF level in bulk cells with 87-90% on-target allele modifications) and reduced sodium metabisulfite induced sickling (from 84% to 29% of erythrocytes). Base editing of non-mobilized PB CD34+ HSPCs from 3 beta-thalassemia patient donors potently induced gamma-globin and improved ineffective erythropoiesis in vitro, with 59-75% on-target allele modifications. Moreover highly efficient multiplex editing could be achieved in HSPCs. Simultaneous disruption of the BCL11A +58 erythroid enhancer core GATA1 motif and correction of the HBB -28A>G promoter mutation in HSPCs resulted in production of at least one therapeutic allele in 35 of 35 erythroid colonies analyzed, with each colony achieving improved alpha-to-non-alpha globin chain balance. Finally we found that base editing could be efficiently produced in engrafting HSCs. Using 3 donors, we observed 62-64% base edits at the BCL11A +58 enhancer GATA1 motif in input HSPCs with one round of electroporation and 73-85% base edits with two rounds of electroporation (separated by 24 hours), albeit with modest reduction of cell viability with tandem electroporation. We observed 70-94% human chimerism and 35-82% base edits in engrafting human cells in primary recipients and 11-61% base edits in secondary recipients as measured 16 weeks following cell infusion. Similar edit frequencies were observed across all assayed hematopoietic lineages including engrafting B-lymphocytes, erythroid precursors, and HSPCs. We observed that compared to non-engrafting progenitors, long-term engrafting HSCs favored C to T (as compared to C to G/A) editing. There was a strong correlation (Spearman r 0.86, P Disclosures No relevant conflicts of interest to declare.

Nonhomologous end-joining (NHEJ) is a key pathway for efficient repair of DNA double-strand breaks (DSBs) and V(D)J recombination. NHEJ defects in humans cause immunodeficiency and increased cellular sensitivity to ionizing irradiation (IR) and are variably associated with growth retardation, microcephaly, and neurodevelopmental delay. Repair of DNA DSBs is important for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). To compare the specific contribution of DNA ligase 4 (LIG4), Artemis, and DNA-protein kinase catalytic subunit (PKcs) in this process and to gain insights into phenotypic variability associated with these disorders, we reprogrammed patient-derived fibroblast cell lines with NHEJ defects. Deficiencies of LIG4 and of DNA-PK catalytic activity, but not Artemis deficiency, were associated with markedly reduced reprogramming efficiency, which could be partially rescued by genetic complementation. Moreover, we identified increased genomic instability in LIG4-deficient iPSCs. Cell cycle synchronization revealed a severe defect of DNA repair and a G0/G1 cell cycle arrest, particularly in LIG4- and DNA-PK catalytically deficient iPSCs. Impaired myeloid differentiation was observed in LIG4-, but not Artemis- or DNA-PK-mutated iPSCs. These results indicate a critical importance of the NHEJ pathway for somatic cell reprogramming, with a major role for LIG4 and DNA-PKcs and a minor, if any, for Artemis.

Progenitor B cells reside in complex bone marrow (BM) microenvironments where they receive signals for growth and maturation. We reported previously that the CXCL12-focal adhesion kinase (FAK)-VLA4 pathway plays an important role in progenitor B cell adhesion and migration. In this study, we have conditionally targeted in B cells FAK, and found that the numbers of progenitor pro-B, pre-B, and immature B cells are reduced by 30–40% in B cell-specific FAK knockout mice. When cultured in methylcellulose with IL-7 ± CXCL12, Fak-deleted pro-B cells yield significantly fewer cells and colonies. Using in situ quantitative imaging cytometry, we establish that in longitudinal femoral BM sections, pro-B cells are preferentially localized in close proximity to the endosteum of the metaphyses and the diaphysis. Fak deletion disrupts the nonrandom distribution of pro-B cells and induces the mobilization of pro-B cells to the periphery in vivo. These effects of Fak deletion on pro-B cell mobilization and localization in BM are amplified under inflammatory stress, that is, after immunization with nitrophenol-conjugated chicken γ-globulin in alum. Collectively, these studies suggest the importance of FAK in regulating pro-B cell homeostasis and maintenance of their spatial distribution in BM niches.

A proportion of homologous recombination (HR) events in mammalian cells resolve by “long tract” gene conversion, reflecting copying of several kilobases from the donor sister chromatid prior to termination. Cells lacking the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, or certain other HR-defective cells, reveal a bias in favor of long tract gene conversion, suggesting that this aberrant HR outcome might be connected with genomic instability. If termination of gene conversion occurs in regions lacking homology with the second end of the break, the normal mechanism of HR termination by annealing (i.e., homologous pairing) is not available and termination must occur by as yet poorly defined non-canonical mechanisms. Here we use a previously described HR reporter to analyze mechanisms of non-canonical termination of long tract gene conversion in mammalian cells. We find that non-canonical HR termination can occur in the absence of the classical non-homologous end joining gene XRCC4. We observe obligatory use of microhomology (MH)-mediated end joining and/or nucleotide addition during rejoining with the second end of the break. Notably, non-canonical HR termination is associated with complex breakpoints. We identify roles for homology-mediated template switching and, potentially, MH-mediated template switching/microhomology-mediated break-induced replication, in the formation of complex breakpoints at sites of non-canonical HR termination. This work identifies non-canonical HR termination as a potential contributor to genomic instability and to the formation of complex breakpoints in cancer., Author Summary Complex breakpoints are a recognized feature of cancer genome rearrangements, but the mechanisms that lead to their formation are undefined. Although homologous recombination (HR) is considered a potentially error-free pathway, cells lacking critical HR genes, such as the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, frequently engage error-prone homologous recombination mechanisms in which HR termination does not occur in a timely fashion. We show here that aberrant termination of HR in mammalian cells involves the use of error-prone alternative end joining mechanisms and can lead to the formation of complex breakpoints by means of template switching mechanisms. This suggests that defective termination of homologous recombination underlies some of the complex breakpoints observed in cancer cells.

To be added., Immunoglobulin heavy chain (IgH) class-switch recombination (CSR) replaces initially expressed Cμ (IgM) constant regions (CH) exons with downstream CH exons. Stimulation of B cells with anti-CD40 plus interleukin-4 induces CSR from Cμ to Cγ1 (IgG1) and Cε (IgE), the latter of which contributes to the pathogenesis of atopic diseases. Although Cε CSR can occur directly from Cμ, most mature peripheral B cells undergo CSR to Cε indirectly, namely from Cμ to Cγ1, and subsequently to Cε. Physiological mechanisms that influence CSR to Cγ1 versus Cε are incompletely understood. In this study, we report a role for B cell developmental maturity in IgE CSR. Based in part on a novel flow cytometric IgE CSR assay, we show that immature B cells preferentially switch to IgE versus IgG1 through a mechanism involving increased direct CSR from Cμ to Cε. Our findings suggest that IgE dysregulation in certain immunodeficiencies may be related to impaired B cell maturation.

Autologous gene therapy (GT) for beta-hemoglobinopathies has demonstrated encouraging early safety and efficacy using addition of a sickling-resistant globin gene to stem cells. Another therapeutic strategy for sickle cell disease (SCD) is erythroid-specific inactivation of BCL11A, which is a validated repressor of gamma globin expression (Sankaran et al. Science 2011). This approach has the distinct advantage of simultaneously inducing fetal hemoglobin (HbF) while coordinately decreasing sickle hemoglobin (HbS). Since hemoglobin (Hb) polymerization in sickle red cells is highly dependent on the intracellular concentration of HbS and is strongly inhibited by HbF, effective BCL11A repression should prevent the sickling phenotype within red cells. We have shown that erythroid-specific expression of microRNA-adapted shRNAs (shRNAmiR) targeting BCL11A effectively induces HbF in human erythroid cells derived from transduced HSCs, largely attenuating the hematologic effects of SCD in a murine model while avoiding negative effects in HSCs and B lymphocytes (Brendel et al. JCI 2016). Here we report the initial results of a pilot clinical study utilizing a shRNAmiR lentiviral vector (LVV) targeting BCL11A for autologous GT in SCD patients. Transduction of hematopoietic cells with GMP lentiviral vector (BCH_BB-LCRshRNA(miR)) expressing the shRNAmiR for BCL11A in an erythroid-specific fashion showed no toxicity in engraftment and genotoxicity assays, efficient transduction rates of 80-95% of CD34+-derived erythroid colony forming cells from healthy donors and SCD patients, and >95% of transduced erythroid colonies demonstrating HbF levels of 50-95% of total Hb. Transduction at clinical scale with plerixafor mobilized CD34+ cells from three SCD donors yielded vector copies of 3.7 - 5.2/cell. Patients with severe SCD were screened for eligibility according to an IND enabled, IRB-approved investigator-initiated protocol. The first cohort included patients ≥ 18 years old. After at least 3 months of protocol-required transfusions, autologous CD34+ cells were collected by plerixafor mobilization and apheresis, and then transduced under GMP conditions with the BCH_BB-LCRshRNA(miR) vector. As of July 28, 2018, 3 patients representing the adult cohort had undergone a total of 3 (n=2) or 4 (n=1) days of mobilization. Mean single-day apheresis yields were 3.2 (range 1.5 - 6.8) x 106 CD34+ cells/kg. No Grade 3 or 4 AEs were attributed to mobilization and collection, although one subject developed an incidentally-discovered line-associated atrial clot and pulmonary embolism. Transduced cell products for these 3 patients have cell doses of 3.3 - 6.7 x 106 CD34+ cells/kg, VCN of 3.3 - 5.1 copies per cell and >95% vector-positive CD34+-derived colonies. One subject (BCL002), who had been regularly transfused for 17 years, has undergone infusion of gene-modified cells after myeloablative busulfan conditioning and achieved neutrophil engraftment after 22 days. Post-infusion follow-up is 78 days. At the time of the last analysis 76 days after GT and 64 days after last RBC transfusion (Table 1) subject BCL002 had a sustained Hb of >10 g/dL and, compared to pre-GT, there was a notable absence of irreversibly sickled cells on peripheral smear and a persistently low absolute reticulocyte count consistent with markedly reduced hemolysis. Hb electrophoresis showed 23.3% HbF, 51.8% HbS and 22.3% HbA (from residual transfused cells) with a HbF/(HbF+HbS) ratio of 29.7%. At day 76, the number of F cells had risen to 59.7% with 12pg HbF/F cell. In flow-sorted immature erythroid cells γ-globin mRNA was >80% of total β-like globins in the graft-derived population and BCL11A protein was reduced by ~90%. Adverse events observed from the start of conditioning until latest follow-up were consistent with myeloablative conditioning, and there have been no product-related adverse events and no SCD-related complications. These early results show: (1) feasibility of enrollment, cell procurement, and GMP manufacturing of gene modified CD34+ cells in 3 adult SCD patients; (2) the first proof of principle demonstrating shRNAmiR-based gene knockdown in humans, and (3) successful rapid induction of HbF in the first patient infused, with marked attenuation of hemolysis in the early phase of autologous reconstitution. Based on the trajectory of increasing HbF/(HbF+HbS), near full suppression of the SCD phenotype is expected. Disclosures Esrick: Bluebird Bio: Honoraria. Negre:bluebird bio: Other: Spouse employed by bluebird Bio. Dansereau:Bluebird Bio: Consultancy. Braunewell:Bluebird Bio: Employment, Equity Ownership. Christiansen:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Nikiforow:Kite Pharma: Consultancy. Achebe:Luitpold Pharmaceutical: Consultancy; AMAG Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Syros pharmaceuticals: Consultancy. Negre:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Heeney:Sancilio Pharmaceuticals: Consultancy, Research Funding; Ironwood: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Vertex/Crisper: Other: Data Monitoring Committee; Pfizer: Research Funding; Astra Zeneca: Consultancy, Research Funding. Williams:Bluebird Bio: Research Funding.

Although therapeutic genome editing of autologous hematopoietic stem cells (HSCs) in principle could cure β-hemoglobinopathies, CRISPR-Cas9 mediated gene modification has demonstrated variable efficiency, specificity, and persistence in HSCs. Here we demonstrate selection-free on-target editing of the BCL11A erythroid enhancer by Cas9:sgRNA ribonucleoprotein in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. First we screened a set of 20 guide RNAs targeting the functional core of the +58 BCL11A enhancer for maximal HbF induction by RNP delivery. We used SpCas9 protein with additional NLS sequences, synthetic modified sgRNA, and optimized electroporation buffer to produce >95% on-target indels disrupting a critical GATA1 binding site within the +58 BCL11A enhancer in CD34+ HSPCs. Clonal analysis showed that even 1 bp indels around the cleavage site were sufficient for HbF reactivation. Specificity was evaluated by CIRCLE-seq, a method to define genome-wide target sequences susceptible to RNP cleavage in vitro. Amplicon deep sequencing of 24 possible off-target sites from edited CD34+ cells did not reveal any off-target editing with limit of detection 0.1% allele frequency. Despite transient induction of a p53 transcriptional response peaking at 4-8 hours after RNP electroporation, we found no evidence of selection for TP53 or 94 other hematologic malignancy associated mutations by targeted deep sequencing. Edited CD34+ HSPCs from healthy donors contributed to multilineage engraftment of primary and secondary immunodeficient mouse recipients at similar levels as unedited control cells. Likewise we found that edited CD34+ HSPCs from a plerixafor-mobilized SCD donor contributed to marrow engraftment and multilineage hematopoiesis in immunodeficient NBSGW mice after 16 weeks at similar levels as unedited cells, with ~95% indel frequency for engrafting healthy and SCD donor cells. Edited engrafting SCD cells were similarly competent for secondary transplantation as unedited controls. Erythroid progeny of edited engrafting sickle cell disease HSCs expressed therapeutic levels of fetal hemoglobin (HbF) and resisted sickling. Erythroid progeny of edited CD34+ HSPCs from 7 transfusion-dependent ß-thalassemia donors showed restored globin chain balance and amelioration of microcytosis and poikilocytosis. We found that compared to the bulk CD34+ HSPC pool, HSCs preferentially underwent nonhomologous as compared to microhomology mediated end-joining repair based on three assays: sorting of CD34+ CD38- CD90+ CD45RA- cells; isolation of cells in G0, G1, S, and G2/M cell cycle phases; and evaluation of long-term engrafting cells in immunodeficient mice. Together these data show that NHEJ-based BCL11A enhancer editing approaching complete allelic disruption is a practicable therapeutic strategy to produce durable HbF induction in SCD and ß-thalassemia. Disclosures Esrick: Bluebird Bio: Honoraria. Williams:Bluebird Bio: Research Funding.

During an immune response, B cells undergo rapid proliferation and activation-induced cytidine deaminase (AID)-dependent remodeling of immunoglobulin (IG) genes within germinal centers (GCs) to generate memory B and plasma cells. Unfortunately, the genotoxic stress associated with the GC reaction also promotes most B cell malignancies. Here, we report that exogenous and intrinsic AID-induced DNA strand breaks activate ATM, which signals through an LKB1 intermediate to inactivate CRTC2, a transcriptional coactivator of CREB. Using genome-wide location analysis, we determined that CRTC2 inactivation unexpectedly represses a genetic program that controls GC B cell proliferation, self-renewal, and differentiation while opposing lymphomagenesis. Inhibition of this pathway results in increased GC B cell proliferation, reduced antibody secretion, and impaired terminal differentiation. Multiple distinct pathway disruptions were also identified in human GC B cell lymphoma patient samples. Combined, our data show that CRTC2 inactivation, via physiologic DNA damage response signaling, promotes B cell differentiation in response to genotoxic stress.

Ig heavy chain (IgH) class-switch recombination (CSR) replaces the IgH Cμ constant region exons with one of several sets of downstream IgH constant region exons (e.g., Cγ, Cε, or Cα), which affects switching from IgM to another IgH class (e.g., IgG, IgE, or IgA). Activation-induced cytidine deaminase (AID) initiates CSR by promoting DNA double-strand breaks (DSBs) within switch (S) regions flanking the donor Cμ (Sμ) and a downstream acceptor CH(e.g., Sγ, Sε, Sα) that are then joined to complete CSR. DSBs generated in Sμ frequently are joined within Sμ to form internal switch region deletions (ISD). AID-induced ISD and mutations have been considered rare in downstream S regions, suggesting that AID targeting to these S regions requires its prior recruitment to Sμ. We have now assayed for CSR and ISD in B cells lacking Sμ (Sμ−/−B cells). In Sμ−/−B cells activated for CSR to IgG1 and IgE, CSR to IgG1 was greatly reduced; but, surprisingly, CSR to IgE occurred at nearly normal levels. Moreover, normal B cells had substantial Sγ1 ISD and increased mutations in and near Sγ1, and levels of both were greatly increased in Sμ−/−B cells. Finally, Sμ−/−B cells underwent downstream CSR between Sγ1 and Sε on alleles that lacked Sμ CSR to these sequences. Our findings show that AID targets downstream S regions independently of CSR with Sμ and implicate an alternative pathway for IgE class switching that involves generation and joining of DSBs within two different downstream S regions before Sμ joining.

Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA-specific cytidine deaminase that initiates Ig heavy chain (IgH) class switch recombination (CSR) and Ig somatic hypermutation (SHM) by deaminating cytidines within, respectively, IgH switch (S) regions and Ig variable region (V) exons. AID that is phosphorylated on serine residue 38 interacts with replication protein A (RPA), a ssDNA binding protein, to promote deamination of transcribed double-stranded DNA in vitro, which, along with other evidence, suggests that AID may similarly gain access to transcribed S regions and V exons in vivo. However, the physiological role of AID phosphorylation at serine residue 38 (S38), and even the requirement for the S38 residue, with respect to CSR or SHM has been debated. To address this issue, we used gene targeting to generate an endogenous mouse AID locus that produces AID in which S38 is substituted with alanine (AID S38A ), a mutant form of AID that retains similar catalytic activity on ssDNA as WT AID (AID WT ). B cells homozygous for the AID S38A mutation show substantially impaired CSR and SHM, correlating with inability of AID S38A to interact with endogenous RPA. Moreover, mice haploinsufficient for AID S38A have even more severely impaired CSR when compared with mice haploinsufficient for AID WT , with CSR levels reduced to nearly background levels. These results unequivocally demonstrate that integrity of the AID S38 phosphorylation site is required for normal CSR and SHM in mice and strongly support a role for AID phosphorylation at S38 and RPA interaction in regulating CSR and SHM.

Immunoglobulin (Ig) class switch recombination (CSR) involves the breakage and subsequent repair of two DNA sequences, known as switch (S) regions, which flank IgH constant region exons. The resolution of CSR-associated breaks is thought to require the nonhomologous end-joining (NHEJ) DNA repair pathway, but the role of the NHEJ factor DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in this process has been unclear. A new study, in which broken IgH-containing chromosomes in switching B cells were visualized directly, clearly demonstrated that DNA-PKcs and, unexpectedly, the nuclease Artemis are involved in the resolution of switch breaks.

Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks (DSBs) during V(D)J recombination in developing lymphocytes and during immunoglobulin (Ig) heavy chain (IgH) class switch recombination (CSR) in peripheral B lymphocytes. We now show that CD21-cre-mediated deletion of the Xrcc4 NHEJ gene in p53-deficient peripheral B cells leads to recurrent surface Ig-negative B lymphomas ("CXP lymphomas"). Remarkably, CXP lymphomas arise from peripheral B cells that had attempted both receptor editing (secondary V[D]J recombination of Igkappa and Iglambda light chain genes) and IgH CSR subsequent to Xrcc4 deletion. Correspondingly, CXP tumors frequently harbored a CSR-based reciprocal chromosomal translocation that fused IgH to c-myc, as well as large chromosomal deletions or translocations involving Igkappa or Iglambda, with the latter fusing Iglambda to oncogenes or to IgH. Our findings reveal peripheral B cells that have undergone both editing and CSR and show them to be common progenitors of CXP tumors. Our studies also reveal developmental stage-specific mechanisms of c-myc activation via IgH locus translocations. Thus, Xrcc4/p53-deficient pro-B lymphomas routinely activate c-myc by gene amplification, whereas Xrcc4/p53-deficient peripheral B cell lymphomas routinely ectopically activate a single c-myc copy.

International audience; Molecular mechanisms underlying synapsis of activation-induced deaminase (AID)-targeted S regions during class switch recombination (CSR) are poorly understood. By using chromosome conformation capture techniques, we found that in B cells, the Emicro and 3'Ealpha enhancers were in close spatial proximity, forming a unique chromosomal loop configuration. B cell activation led to recruitment of the germline transcript (GLT) promoters to the Emicro:3'Ealpha complex in a cytokine-dependent fashion. This structure facilitated S-S synapsis because Smicro was proximal to Emicro and a downstream S region was corecruited with the targeted GLT promoter to Emicro:3'Ealpha. We propose that GLT promoter association with the Emicro:3'Ealpha complex creates an architectural scaffolding that promotes S-S synapsis during CSR and that these interactions are stabilized by AID. Thus, the S-S synaptosome is formed as a result of the self-organizing transcription system that regulates GLT expression and may serve to guard against spurious chromosomal translocations.

p53-Binding protein 1 (53BP1) encodes a critical checkpoint protein that localizes to sites of DNA double-strand breaks (DSBs) and participates in DSB repair. Mice that are 53bp1 deficient or hemizygous have an increased incidence of lymphoid malignancies. However, 53BP1 abnormalities in primary human tumors have not been described. By combining high-density single nucleotide polymorphism (HD SNP) array data and gene expression profiles, we found 9 of 63 newly diagnosed human diffuse large B-cell lymphomas (DLBCLs) with single copy loss of the chromosome 15q15 region including the 53BP1 locus; these nine tumors also had significantly lower levels of 53BP1 transcripts. 53BP1 single copy loss found with the HD SNP array platform was subsequently confirmed by fluorescence in situ hybridization. These studies highlight the role of 53BP1 copy loss in primary human DLBCLs and the value of integrative analyses in detecting this genetic lesion in human tumors.

During B cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons1,2. In mice, six additional sets of constant region exons (CHs) lie 100-200 kb downstream in the same transcriptional orientation as V(D)J and Cμ exons2. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH2. Activation-Induced Cytidine Deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region2,3; these lesions are converted into DNA double-strand breaks (DSBs) by general DNA repair factors3. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S region DSB (Fig. 1a). However, the relative frequency of deletional to inversional CSR junctions had not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved was unknown. To address this question, we adapted high-throughput genome-wide translocation sequencing (HTGTS)4 into a highly sensitive DSB end-joining assay and applied it to endogenous AID-initiated S region DSBs. We find that CSR indeed is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis IgH organizational features in combination with frequent S region DSBs initiated by AID. We further implicate ATM-dependent DSB response (DSBR) factors in enforcing this mechanism and provide a solution to the enigma of why CSR is so reliant on the 53BP1 DSBR factor.

Guarding the genome against internal and external assaults requires the coordinated interaction of multiple cellular networks to sense, respond to, and repair breaks in chromosomal DNA. Both external factors such as ionizing radiation or internal events like oxidative damage can cause DNA double stranded breaks (DSBs) [1] , [2] . DSBs are also part of the normal lymphocyte developmental program where they are an integral element of the mechanisms that generate a diverse immune repertoire in the context of V(D)J and immunoglobulin heavy chain (IgH) class switch recombination (CSR) [3] , [4] , [5] . DSBs initiate a cascade of cellular events that direct cells to pause and properly repair potentially lethal chromosomal breaks. Errors in the repair of both general and lymphocyte-specific DSBs can lead to oncogenic chromosomal translocations [6] , [7] , [8] . Here, we review recent advances in understanding factors and protein complexes involved in the response to DNA DSBs with a focus on the B lymphocyte specific process of CSR.

Histone H2AX promotes DNA double-strand break (DSB) repair and immunoglobulin heavy chain (IgH) class switch recombination (CSR) in B-lymphocytes. CSR requires activation-induced cytidine deaminase (AID) and involves joining of DSB intermediates by end joining. We find that AID-dependent IgH locus chromosome breaks occur at high frequency in primary H2AX-deficient B cells activated for CSR and that a substantial proportion of these breaks participate in chromosomal translocations. Moreover, activated B cells deficient for ATM, 53BP1, or MDC1, which interact with H2AX during the DSB response, show similarly increased IgH locus breaks and translocations. Thus, our findings implicate a general role for these factors in promoting end joining and thereby preventing DSBs from progressing into chromosomal breaks and translocations. As cellular p53 status does not markedly influence the frequency of such events, our results also have implications for how p53 and the DSB response machinery cooperate to suppress generation of lymphomas with oncogenic translocations.