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Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing–based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.

The Primary Immune Deficiency Treatment Consortium (PIDTC) performed a retrospective analysis of 662 patients with severe combined immunodeficiency (SCID) who received a hematopoietic cell transplantation (HCT) as first-line treatment between 1982 and 2012 in 33 North American institutions. Overall survival was higher after HCT from matched-sibling donors (MSDs). Among recipients of non-MSD HCT, multivariate analysis showed that the SCID genotype strongly influenced survival and immune reconstitution. Overall survival was similar for patients with RAG, IL2RG, or JAK3 defects and was significantly better compared with patients with ADA or DCLRE1C mutations. Patients with RAG or DCLRE1C mutations had poorer immune reconstitution than other genotypes. Although survival did not correlate with the type of conditioning regimen, recipients of reduced-intensity or myeloablative conditioning had a lower incidence of treatment failure and better T- and B-cell reconstitution, but a higher risk for graft-versus-host disease, compared with those receiving no conditioning or immunosuppression only. Infection-free status and younger age at HCT were associated with improved survival. Typical SCID, leaky SCID, and Omenn syndrome had similar outcomes. Landmark analysis identified CD4(+) and CD4(+)CD45RA(+) cell counts at 6 and 12 months post-HCT as biomarkers predictive of overall survival and long-term T-cell reconstitution. Our data emphasize the need for patient-tailored treatment strategies depending upon the underlying SCID genotype. The prognostic significance of CD4(+) cell counts as early as 6 months after HCT emphasizes the importance of close follow-up of immune reconstitution to identify patients who may need additional intervention to prevent poor long-term outcome.

Background Sickle cell disease (SCD) is a recessive monogenic disease caused by a single point mutation in which glutamic acid replaces valine in Codon 6 of the human beta-globin gene (HBB) leading to the production of abnormal globin chains (HbS) that polymerize and cause erythrocytes to sickle. This results in hemolytic anemia, vaso-occlusion and organ damage, which leads to lifelong complications and early mortality. Allogeneic hematopoietic stem cell transplant (allo-HSCT) is the only known cure for SCD, however, its use is limited by the lack of well-matched donors, need for immunosuppression, risk of graft versus host disease and graft rejection. GPH101 is an investigational, autologous, hematopoietic stem cell (HSC) drug product (DP) designed to correct the SCD mutation in the HBB gene ex vivo using a high fidelity Cas9 (CRISPR associated protein 9) paired with an AAV6 (adeno-associated virus type 6) delivery template, efficiently harnessing the natural homology directed repair (HDR) cellular pathway. This approach has the potential to restore normal adult hemoglobin (HbA) production while simultaneously reducing HbS levels. In preclinical studies, HBB gene correction in SCD donor HSCs resulted in ≥60% of gene-corrected alleles in vitro with minimal off-target effects. Gene corrected cells were successfully differentiated toward the erythroid lineage and produced ≥70% HbA in vitro. Long-term engraftment of gene-corrected HSCs was demonstrated in vivo, following transplant into immunodeficient mice, with multi-lineage allelic gene correction frequencies well above the predicted curative threshold of 20%, with potential of this approach to be equivalent or superior to allo-HSCT. In addition, HSC-based correction in an SCD mouse model led to stable adult hemoglobin production, increased erythrocyte lifespan and reduction in sickling morphology, demonstrating the therapeutic potential of this gene correction platform as a curative approach in SCD. Study Design and Methods CEDAR (NCT04819841) is a first-in-human, open-label, single-dose, multi-site Phase 1/2 clinical trial in participants with severe SCD designed to evaluate safety, efficacy and pharmacodynamics (PD) of GPH101. Approximately 15 adult (18-40 years) and adolescent (12-17 years) participants will be enrolled across 5 sites, with adolescent enrollment proceeding after a favorable assessment of adult safety data by a Safety Monitoring Committee. Participants must have a diagnosis of severe SCD (βS/βS), defined as ≥ 4 severe vaso-occlusive crises (VOCs) in the 2 years prior and/or ≥ 2 episodes of acute chest syndrome (ACS), in 2 years prior with at least 1 episode in the past year. Participants on chronic transfusion therapy may be eligible if required VOC and ACS criteria are met in the 2 years prior to the initiation of transfusions. Key exclusion criteria include availability of a 10/10 human leukocyte antigen-matched sibling donor, or prior receipt of HSCT or gene therapy. After eligibility confirmation including screening for pre-treatment cytogenetic abnormalities, participants will undergo plerixafor mobilization and apheresis, followed by CD34+ cell enrichment and cryopreservation, undertaken locally at each trial site before shipment to a centralized manufacturer for GPH101 production. After GPH101 release, participants will undergo eligibility reconfirmation prior to busulfan conditioning and DP infusion. Safety, efficacy and PD measurements will occur for 2 years post-infusion; a long-term follow up study will be offered to participants for an additional 13 years of monitoring. The primary endpoint for this study is safety, measured by the kinetics of HSC engraftment, transplant related mortality, overall survival and frequency and severity of adverse events. Secondary endpoints will explore efficacy and PD, including levels of globin expression as compared to baseline, gene correction rates, clinical manifestations of SCD (including VOC and ACS), laboratory parameters, complications and organ function. In addition, cerebral hemodynamics and oxygen delivery will be assessed by magnetic resonance techniques. Key exploratory endpoints include evaluation of patient-reported outcomes, erythrocyte function, on-target and off-target editing rates, and change from baseline in select SCD characteristics. Disclosures Kanter: Fulcrum Therapeutics, Inc.: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Forma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees; Beam: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Graphite Bio: Consultancy; GuidePoint Global: Honoraria; Fulcrum Tx: Consultancy. Thompson: Agios Pharmaceuticals: Consultancy; Graphite Bio: Research Funding; Vertex: Research Funding; Beam Therapeutics: Consultancy; Celgene: Consultancy, Research Funding; Biomarin: Research Funding; Baxalta: Research Funding; CRISPR Therapeutics: Research Funding; Global Blood Therapeutics: Current equity holder in publicly-traded company; bluebird bio: Consultancy, Research Funding; Novartis: Research Funding. Porteus: Versant Ventures: Consultancy; CRISPR Therapeutics: Current equity holder in publicly-traded company; Allogene Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Ziopharm: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Graphite Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Intondi: Graphite Bio: Current Employment, Current equity holder in publicly-traded company; Global Blood Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Lahiri: Graphite Bio: Current Employment, Current equity holder in publicly-traded company. Dever: Graphite Bio: Current Employment, Current equity holder in publicly-traded company. Petrusich: bluebird bio: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Graphite Bio: Current Employment, Current equity holder in publicly-traded company. Lehrer-Graiwer: Global Blood Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Graphite Bio: Current Employment, Current equity holder in publicly-traded company.

Reticular Dysgenesis (RD) is a particularly grave form of severe combined immunodeficiency (SCID), characterized by maturation arrest of both myeloid and lymphoid lineages paired with sensorineural hearing loss. RD is caused by biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2). AK2 catalyzes the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) in the mitochondrial intermembrane space. Using a CRISPR/Cas9 AK2 biallelic knock out model in human hematopoietic stem and progenitor cells (HSPCs), we have shown that AK2 -/- HSPCs mimic the neutrophil maturation defect in RD patients. Mitochondrial respiration is compromised in AK2 -/- HSPCs, which leads to a decreased NAD +/NADH ratio resulting in reductive stress. Metabolomics analysis by LC-MS/MS showed a significant accumulation of AMP, along with increased AMP/ADP and AMP/ATP ratios in AK2 -/- HSPCs, suggesting that purine metabolism is compromised by AK2 deficiency. Purine metabolism defects, such as deficiencies in adenosine deaminase (ADA) and purine nucleotide phosphorylase (PNP), have long been recognized as a cause of SCID. Furthermore, pharmacological interference with purine metabolism is a highly effective antiproliferative strategy in cancer therapy. In this study, we sought to investigate whether impaired purine metabolism contributes to the myelopoietic defect caused by AK2 deficiency. Results We explored how purine metabolism affects myelopoiesis by differentiating HSPCs in media containing no nucleosides (nucleoside-), mixed nucleosides (nucleoside+) or adenosine only (adenosine+). We observed no difference in proliferation or neutrophil maturation between nucleosides- and nucleoside+ media for both control and AK2 -/- HSPCs, suggesting that AK2 -/- HSPCs do not rely on exogenous nucleosides. Interestingly, control HSPCs cultured in adenosine+ media showed severe proliferation and neutrophil maturation defects that mimic AK2 deficiency, suggesting that purine imbalance is detrimental to myelopoiesis. Previous metabolomics analysis showed a significant accumulation of inosine monophosphate (IMP) in AK2 -/- HSPCs. Since IMP can be produced through AMP deamination by AMPD, we asked whether the IMP accumulation in AK2 -/- HSPCs is caused by converting excess AMP to IMP. An LC-MS/MS analysis showed that AMPD inhibitor (AMPDi) treatment significantly lowered IMP levels and increased AMP levels in AK2 -/- HSPCs, indicating that AMP deamination is a major source of IMP accumulation in AK2 -/- HSPCs. Furthermore, AMPDi treatment did not improve, but rather slightly aggravated neutrophil differentiation in AK2 -/- HSPCs, suggesting that the AK2 -/- neutrophil maturation defect is not caused by IMP accumulation. This raises the possibility that AK2 -/- HSPCs employ AMP deamination as a mechanism to curtail the toxicity of excess AMP. Since purine is a building block of RNA, and ribosomal RNA (rRNA) constitutes >85% of cellular RNA content, we asked whether rRNA synthesis is compromised by AK2 deficiency. Pyronin Y staining showed a significant decrease in rRNA content in AK2 -/- HSPCs. Nascent peptide synthesis rate was also decreased in AK2 -/- HSPCs, as quantified by OP-puromycin uptake. These findings are corroborated by RNA-seq analysis of AK2 -/- and control HSPCs, which showed that ribosomal subunits, ribosomal biogenesis and ribonucleoprotein complex assembly are among the top down-regulated pathways. The data suggest that defective purine metabolism in AK2 -/- HSPCs impairs ribosomal biogenesis and protein synthesis. Conclusion Our studies showed that purine imbalance in HSPCs impairs myeloid proliferation and neutrophil maturation. AK2 depletion in HSPCs leads to AMP accumulation and defective ribosomal biogenesis. AK2 -/- HSPCs convert excess AMP to IMP, possibly as a means to mitigate AMP toxicity. However, AMP deamination activities alone are not sufficient to lower AMP levels to those of control HSPCs. We are currently testing whether boosting 5'-nucleotidase activities (cNIA, cN1B and cNII) in AK2 -/- HSPCs can decrease AMP levels and rescue the neutrophil maturation defect. As purine metabolic defects are associated with diverse immune and non-immune abnormalities, further understanding of how purine metabolism governs differentiation of human HSPCs will enable us to develop novel therapeutic strategies for RD and other purine disorders. Disclosures Porteus: CRISPR Therapeutics: Current equity holder in publicly-traded company; Allogene Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Versant Ventures: Consultancy; Ziopharm: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Graphite Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Morrison: Garuda Therapeutics: Other: founder and SAB member ; Kojin Therapeutics: Other: SAB member ; Frequency Therapeutics: Other: SAB member ; Ona Terapeutics: Other: SAB member ; Protein Fluidics: Other: SAB member .

Gene editing is a rapidly developing area of biotechnology in which the nucleotide sequence of the genome of living cells is precisely changed. The use of genome-editing technologies to modify various types of blood cells, including hematopoietic stem cells, has emerged as an important field of therapeutic development for hematopoietic disease. Although these technologies offer the potential for generation of transformative therapies for patients suffering from myriad disorders of hematopoiesis, their application for therapeutic modification of primary human cells is still in its infancy. Consequently, development of ethical and regulatory frameworks that ensure their safe and effective use is an increasingly important consideration. Here, we review a number of issues that have the potential to impact the clinical implementation of genome-editing technologies, and suggest paths forward for resolving them such that new therapies can be safely and rapidly translated to the clinic.

Energy deficiency and redox stress are hallmarks of mitochondrial pathology. Reductive stress is marked by an accumulation of reducing species and can arise from defects in the electron transport chain (ETC) that prevent NAD+ regeneration from NADH. Reticular Dysgenesis (RD) is a particularly grave form of severe combined immunodeficiency (SCID), characterized by maturation arrest of both myeloid and lymphoid lineages. Unlike other forms of SCID, RD is a mitochondriopathy caused by biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2). AK2 catalyzes the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP), and maintains ADP availability for ATP synthase. We hypothesize that AK2 deficiency leads to decreased ETC activities and defective NAD+ regeneration. Emerging evidence suggests that the development of hematopoietic stem and progenitor cells (HSPCs) is intricately intertwined with various aspects of mitochondrial function. Investigating the cellular and molecular consequences of AK2 deficiency during myelopoiesis provides fundamental insight into the pathology of many mitochondrial disorders. Methods: To recapitulate RD myeloid maturation defects, we developed an AK2 biallelic knock out model in human HSPCs using CRISPR gene editing. HSPCs were edited at the AK2 locus, and cells with biallelic AK2 knock out were enriched using homologous recombination-mediated dual reporters. HSPCs edited at the safe harbor locus AAVS1 were used as a control. When differentiated along the myeloid lineage in vitro, AK2-/- HSPCs showed significantly decreased proliferation, lower commitment to the granulocytic lineage, and maturation arrest at the promyelocyte stage, mimicking the presentation of RD patients. To dissect differentiation stage specific changes in metabolism, metabolomics analysis (LC-MS/MS), metabolic flux analysis (Seahorse assays) and RNA-seq were performed on FACS sorted populations of promyelocytes (PMs), metamyelocytes (MCs) and neutrophils (NPs). Additionally, mitochondrial membrane potential and ribosomal RNA (rRNA) content were quantified using TMRM and pyronin Y staining. Results: AK2-/- MCs and NPs showed higher AMP levels, and increased AMP/ADP and AMP/ATP ratios, in line with AK2's function to regenerate ADP from AMP. Mitochondrial oxygen consumption rate decreased, and mitochondrial membrane potential increased in AK2-/- MCs and NPs, indicating defective ETC function and ATP synthesis. Consistent with these results, TCA cycle metabolites were downregulated while pathways that fuel the TCA cycle, i.e. glycolysis and fatty acid oxidation, were upregulated. Interestingly, we observed a significant decrease in NAD+ levels, and an increase in NADH/NAD+ and GSH/GSSG ratios in AK2-/- MCs and NPs, indicative of reductive stress. These results suggest that AK2 deficiency compromises mitochondrial respiration, leading to NAD+ depletion and reductive stress in later stages of myeloid development. Defective mitochondrial respiration has been shown to impair NAD+-dependent aspartate and purine biosynthesis. In AK2-/- MCs and NPs, we observed a profound aspartate depletion and build-up of the purine precursor inosine monophosphate (IMP). As a building block for DNA and RNA, purine deficiency is known to block cell proliferation. Genes in cell cycle and ribosomal biogenesis pathways were down regulated in AK2-/- MCs and NPs. In addition, rRNA content was significantly decreased. These data raise the possibility that purine deficiency in AK2-/- HSPCs compromises nucleotide/protein synthesis along with cell cycle progression. Conclusions: Using an AK2 biallelic knock out HSPC model for RD, we have shown that defective mitochondrial respiration in AK2-/- HSPCs leads to reductive stress, NAD+ and purine depletion resulting in compromised nucleotide/protein synthesis and impaired cell cycle progression. Notably, these defects worsen as myeloid maturation progresses, possibly reflecting the increasing mitochondrial metabolic demand. We are currently exploring whether correcting the NADH/NAD+ ratio in AK2-/- HSPCs improves purine synthesis and restores myelopoiesis. Understanding how redox metabolism governs HSPC differentiation will not only allow us to delineate metabolic changes during development, but enable us to develop novel therapies for RD and other mitochondrial disorders. Disclosures Dever: Integral Medicines: Current Employment.

Loss of CD40 ligand (CD40L) expression or function results in X-linked hyper-immunoglobulin (Ig)M syndrome (X-HIGM), characterized by recurrent infections due to impaired immunoglobulin class-switching and somatic hypermutation. Previous attempts using retroviral gene transfer to correct murine CD40L expression restored immune function; however, treated mice developed lymphoproliferative disease, likely due to viral-promoter-dependent constitutive CD40L expression. These observations highlight the importance of preserving endogenous gene regulation in order to safely correct this disorder. Here, we report efficient, on-target, homology-directed repair (HDR) editing of the CD40LG locus in primary human T cells using a combination of a transcription activator-like effector nuclease-induced double-strand break and a donor template delivered by recombinant adeno-associated virus. HDR-mediated insertion of a coding sequence (green fluorescent protein or CD40L) upstream of the translation start site within exon 1 allowed transgene expression to be regulated by endogenous CD40LG promoter/enhancer elements. Additionally, inclusion of the CD40LG 3'-untranslated region in the transgene preserved posttranscriptional regulation. Expression kinetics of the transgene paralleled that of endogenous CD40L in unedited T cells, both at rest and in response to T-cell stimulation. The use of this method to edit X-HIGM patient T cells restored normal expression of CD40L and CD40-murine IgG Fc fusion protein (CD40-muIg) binding, and rescued IgG class switching of naive B cells in vitro. These results demonstrate the feasibility of engineered nuclease-directed gene repair to restore endogenously regulated CD40L, and the potential for its use in T-cell therapy for X-HIGM syndrome.

While hematopoietic stem and progenitor cells (HSPCs) were thought to rely mainly on glycolysis for energy supply, emerging evidence suggests that defects in mitochondrial functions can impact HSPC development with respect to self-renewal, differentiation and aging. The exact mechanisms underlying metabolic reprogramming and cell fate decisions during human hematopoiesis, however, remain elusive. Biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2), cause reticular dysgenesis (RD), one of the most profound forms of severe combined immunodeficiency (SCID). AK2 catalyzes the interconversion between adenine nucleotides and thereby controls the availability of ADP for oxidative phosphorylation. Clinically, RD patients not only present with profound lymphopenia, typical for classic SCID, but also suffer from severe congenital neutropenia. The developmental arrest across the T, NK and granulocytic lineages suggests that AK2 deficiency causes a metabolic defect with global impact on hematopoiesis. Our prior work in induced pluripotent stem cells (iPSCs) from RD patients has shown that maturation-arrested iPSC-derived HSPCs exhibit increased oxidative stress and an energy-depleted adenine nucleotide profile, suggesting that AK2-regulated mitochondrial bioenergetics play an integral role in HSPC differentiation. Therefore, RD serves as an excellent model to study the impact of mitochondrial metabolism during human HSPC development. Methods: Since iPSCs do not recapitulate definitive hematopoiesis, we developed an AK2 biallelic knock-out model in primary human HSPCs using CRISPR/Cas9 gene editing. Employing a homologous recombination-mediated dual color reporter strategy, we were able to select for HSPCs with biallelic AK2 knock-out. HSPCs edited at the safe harbor AAVS1 site were used as a control. FACS purified AK2-/- and AAVS1-/- HSPCs were in vitro differentiated along the granulocytic lineage, and cells at various differentiation stages were sorted for RNA-seq and metabolomics analysis. Results: We analyzed the myeloid differentiation potential of AK2-/- HSPCs in vitro. Compared to AAVS1-/- controls, AK2-/- HSPCs displayed a severely decreased colony forming potential of both myeloid and erythroid lineages. In addition, AK2-/- HSPCs showed a granulocytic maturation arrest at the HLA-DR-, CD117+ promyelocyte stage, consistent with the characteristic phenotype observed in RD patients. We then performed RNA-seq studies on in vitro differentiated promyelocyte and neutrophil subpopulations derived from AK2-/- and control HSPCs. The RNA-seq analysis showed differential gene expression in glutathione metabolism and IL-10 signaling pathways, suggesting an increase in oxidative stress and inflammation, respectively, caused by AK2 deficiency. In addition, genes implicated in antimicrobial function and granule synthesis were downregulated in AK2-/- neutrophils, suggesting a functional defect. Liquid chromatography-mass spectrometry (LC-MS/MS) studies to delineate differences in metabolite profile conferred by AK2 deficiency at different stages of HSPC development are currently in progress. Conclusions: We have established the first cell-traceable biallelic AK2 CRISPR knock-out model in primary human HSPCs that recapitulates the myeloid phenotype of RD patients. This model allows us to profile AK2 knock-out cells at different developmental stages. AK2-/- granulocyte precursors showed a transcriptional signature suggestive of worsening oxidative stress, inflammation and defective effector cell functions during maturation. To understand the mechanistic underpinnings for these observations we are now using a global metabolomics approach to profile the changes in energy metabolites that occur during development in AK2-deficient and control HSPC subpopulations. Understanding how metabolism governs differentiation and self-renewal of human HSPCs has important translational implications to improve hematopoietic stem cell products and transplantation outcomes. Disclosures Morrison: Frequency Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; OncoMed Pharmaceuticals: Equity Ownership; GI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kolon Gene Therapeutics: Consultancy; Protein Fluidics: Other: Stock Options.

Sickle cell disease (SCD) is an inherited blood disorder associated with a debilitating chronic illness. SCD is caused by a point mutation in the β-globin gene (HBB). A single nucleotide substitution converts glutamic acid to a valine that leads to the production of sickle hemoglobin (HbS), which impairs the function of red blood cells. Here we show that delivery of Streptococcus pyogenes (Sp) Cas9 protein and CRISPR guide RNA as a ribonucleoprotein complex (RNP) together with a short single-stranded DNA donor (ssODN) template into CD34+ hematopoietic stem and progenitor cells (HSPCs) from SCD patients' bone marrow (BM) was able to correct the sickling HBB mutation, with up to 33% homology directed repair (HDR) without selection. Further, CRISPR/Cas9 cutting of HBB in SCD HSPCs induced gene conversion between the HBB sequences in the vicinity of the target locus and the homologous region in δ-globin gene (HBD), with up to 4.4% additional gene correction mediated by the HBD conversion in cells with Cas9 cutting only. The erythrocytes derived from gene-edited cells showed a marked reduction of the HbS level, increased expression of normal adult hemoglobin (HbA), and a complete loss of cell sickling, demonstrating the potential in curing SCD. We performed extensive off-target analysis of gene-edited SCD HSPCs using the in-silico prediction tool COSMID and unbiased, genome-wide assay Guide-Seq, revealing a gross intrachromosomal rearrangement event between the on- and off-target Cas9 cutting sites. We used a droplet digital PCR assay to quantify deletion and inversion events from Day 2 to Day 12 after RNP delivery, and found that large chromosomal deletion decreased from 1.8% to 0.2%, while chromosomal inversion maintained at 3.3%. We demonstrated that the use of high-fidelity SpCas9 (HiFi Cas9 by IDT) significantly reduced off-target effects and completely eliminated the intrachromosome rearrangement events, while maintaining the same level of on-target gene editing, leading to high-efficiency gene correction with increased specificity. In order to determine if gene-corrected SCD HSPCs retain the ability to engraft, CD34+ cells from the BM of SCD patients were treated with Cas9/gRNA RNP and ssODN donor for HBB gene correction, cryopreserved at Day 2 post genome editing, then intravenously transplanted into NSG mice shortly after thawing. These mice were euthanized at Week 16 after transplantation, and the BM was harvested to determine the engraftment potential. An average of 7.5 ±5.4% of cells were double positive for HLA and hCD45 in mice injected with gene-edited CD34+ cells, compared to 16.8 ±9.3% with control CD34+ cells, indicating a good level of engraftment of gene-corrected SCD HSPCs. A higher fraction of human cells were positive for CD19 (66 ±28%), demonstrating lymphoid lineage bias. DNA was extracted from unsorted cells, CD19 or CD33 sorted cells for gene-editing analysis; the HBB editing rates were respectively 29.8% HDR, 2.4% HBD conversion, and 42.8% non-homologous end joining (NHEJ) pretransplantation, and editing rates at Week 16 posttransplantation were respectively 8.8 ±12% HDR, 1.8 ±1.7% HBD conversion, and 24.5 ±12% NHEJ. The highly variable editing rate and indel diversity in gene-edited cells at Week 16 in all four transplanted mice suggest clonal dominance of a limited number of HSPCs after transplantation. Taken together, our results demonstrate highly efficient gene and phenotype correction of the sickling mutation in BM HSPCs from SCD patients mediated by HDR and HBD conversion, and the ability of gene-edited SCD HSPCs to engraft in vivo. We also demonstrate the importance of genome-wide analysis for off-target analysis and the use of HiFi Cas9. Our results provide further evidence for the potential of moving genome editing-based SCD treatment into clinical practice. Acknowledgments: This work was supported by the Cancer Prevention and Research Institute of Texas grants RR140081 and RP170721 (to G. B.), and the National Heart, Lung and Blood Institute of NIH (1K08DK110448 to V.S.) Disclosures Porteus: CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Hematopoietic stem cell (HSC) differentiation is accompanied by a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) to meet the increasing energy demand during proliferation and differentiation. However, the role of mitochondrial metabolism in HSC differentiation goes beyond ATP production. Metabolites generated during mitochondrial metabolism may impact in HSC fate decisions through stable epigenetic modifications. Despite some progress in understanding mitochondrial communication during HSC development, their role in human hematopoiesis remains largely elusive, where the lack of appropriate model systems poses a major obstacle. Reticular Dysgenesis (RD), a rare and particularly severe form of severe combined immunodeficiency (SCID), offers an attractive model for studying the role of mitochondrial metabolism in hematopoiesis. RD is an autosomal recessive disease caused by biallelic mutations of the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 catalyzes the reversible phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP), which serves as the substrate for the ATP synthase. In addition to defective lymphocyte development typical of classic SCID, RD patients also suffer from impaired myeloid development, suggestive of a global defect in hematopoiesis. In a human induced pluripotent stem cell (iPSC) model for RD, hematopoietic stem and progenitor cells (HSPCs) recapitulate a profound maturation arrest of the myeloid lineage, increased oxidative stress and an energy-depleted metabolite and transcriptional profile. We hypothesize that AK2 defects drive hematopoietic cell fate decisions through changes in metabolites that regulate the activities of DNA/histone modifying enzymes and result in stable epigenetic modifications. Methods: Since iPSCs are not suitable to model the epigenetic characteristics of definitive hematopoiesis, we developed a novel model system in which we deleted AK2 in primary human HSCs using CRISPR/Cas9 gene editing technique. We found a highly effective single-guide RNA (sgRNA) targeting the catalytic LID domain of the AK2 gene to introduce directed DNA double stranded breaks (DSBs), and use a homologous recombination (HR)-mediated dual reporter system to track and isolate cells with biallelic AK2 disruption. Results: Our single-color GFP reporter system consistently produces a >60% GFP+ population of AK2-targeted CD34+ umbilical cord blood (UCB) cells. With dual GFP/BFP reporters, we were able to achieve 6% GFP/BFP double positive cells with confirmed biallelic AK2 knock-out. Since HR events on one allele are biologically linked to CRISPR/Cas9 mediated DSBs on the other, we assessed insertion and/or deletion (INDEL) frequency and protein expression in a single reporter (GFP+) population of AK2-targeted UCBs. We detected an INDEL frequency of over 90% on the non-HR alleles along with nearly absent AK2 protein expression by Western Blot. These results indicated that the highly efficient single-color reporter system with >60% targeting efficiency is sufficient to achieve an AK2 biallelic knock-out population in primary HSCs. in vitro myeloid differentiation of these AK2-targeted HSCs recapitulates the RD phenotype with impaired neutrophil but preserved monocyte development. Conclusion: This novel disease model for RD will now allow us to examine the cellular and molecular impact of perturbations in metabolism on human HSC development. We will investigate the effect on differentiation potential, metabolite profile, transcriptome and epigenome in vitro as well as in a xenograft mouse model. Elucidating how metabolism governs differentiation and self-renewal of human HSCs will not only advance our basic understanding of many blood and immune diseases, but has important translational implications for improving the use of HSCs in hematopoietic stem cell transplantation, gene and cell therapy. Disclosures Porteus: CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. The disease typically presents within the first year of life. Approximately 70% of DBA patients possess a mutations in one of at least 12 ribosomal proteins, with RPS19 and RPL11 being the most prevalent, accounting for over 25% and 5% of cases respectively. Current therapies for DBA have undesirable side effects, including iron overload from repeated transfusions or infections from immunosuppressive drugs and stem cell transplantation. Nemo-like Kinase (NLK) is chronically hyper-activated in RPS19- and RPL11-haploinsufficient murine and human models of DBA, as well as erythroid progenitors from DBA patients. In an RPS19-insufficient human model, genetic silencing of NLK (shRNA) increased erythroid expansion by 2.2 fold, indicating aberrant NLK activation contributes to the pathogenesis of the disease. In an independent, high-throughput kinase inhibitor screen examining progenitor expansion in RPS19-insufficient Kit+ murine cells, a number of compounds were identified that increased progenitor expansion. SB431542 and SD208 are recognized TGFβ inhibitor compounds, but were the only TGFβ inhibitors of a panel of 11 that increased progenitor expansion. Both active compounds robustly inhibited NLK activity in vitro and in vivo while the remaining 9 inhibitors had no significant impact on NLK. Both compounds increased erythroid expansion in murine (3.1 and 5.4 fold) and human (3.2 and 6.3 fold) models of DBA with no effect on wild type erythropoiesis (EC50 5 µM and 0.7 µM). No further increase in erythroid expansion was observed when NLK expression was silenced by shRNA. Virtually identical results were observed in CD34+ progenitors from 3 DBA patient bone marrow aspirates with 2.3, 1.9 and 2.1 fold increases in CD235+ erythroblast generation compared to untreated. NLK hyperactivation was limited to differentiating committed erythroid progenitors and was not detected in megakaryocytic, other myeloid progenitors or lymphoblastoid cells lines from DBA patients. During differentiation, non-erythroid lineages upregulate miR181, which results in NLK transcript degradation and loss of NLK expression. The absence of NLK in non-erythroid progenitors prevents NLK activation during ribosomal insufficiency. CRISPR/Cas9 mutation of the miR181 binding site in the NLK 3'UTR in RPS19-insufficient CD34+ HSPCs prevented NLK downregulation, increased NLK activity, and sensitized megakaryocyte and other myeloid lineages (80.5% and 76% reduction relative to controls). This is comparable to the erythroid expansion defect in RPS19-insufficiency (80.7% reduction). In differentiating erythroid progenitors, RPS19 insufficiency increased phosphorylation of the mTORC1 component Raptor (5.3 fold), reducing mTOR activity by 82%. This was restored to basal levels upon pharmacological or genetic inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTORC1 activity with leucine has been proposed to increase translational efficiency in DBA patients. Probably due to NLK phosphorylation of raptor, DBA patients did not respond as anticipated. While leucine treatment did increase mTOR activity in both control (100% to 188%) and RPS19-insufficiency (27 % to 42% of control), the combined treatment of leucine with NLK inhibition resulted in increased mTOR activity to 142% of control and significantly improved erythroid expansion. Identification of aberrantly activated enzymes, such as NLK, that are specifically expressed in erythroid progenitors, offer therapeutic promise as potential druggable targets in the clinical management of DBA that can be used in combination with existing therapies. Disclosures Glader: Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Porteus:CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Flygare:LU Holding: Patents & Royalties: Patent.

Pyruvate kinase deficiency (PKD) is the most common erythroid inherited enzymatic defect causing chronic nonspherocytic hemolytic anemia. PKD is an autosomal recessive disorder caused by mutations in the PKLR gene, which led in a total or partial reduction of the activity of the erythroid pyruvate kinase (RPK) protein. To date, more than 200 different mutations in the PKLR gene have been related with PKD. The disease is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. Treatments for PKD are mainly palliative, including regular red blood cell transfusion, splenectomy and iron chelation therapy. Allogeneic hematopoietic stem cell transplant (HSCT) represents the only curative treatment for severely affected patients, so far. Autologous HSCT of genetically corrected cells would offer a durable and curative clinical option. Over the last years, gene editing has emerged as a promising gene therapy approach for blood cell disorders, where genetic alterations can be accurately corrected. The high level of correction got in hematopoietic progenitors and stem cells without remarkable off-target effects suggests that the clinical use of gene editing therapy to correct genetic hematopoietic diseases is highly likely in a short term. Here, we present two gene editing approaches to correct PKD in human hematopoietic cells based of specific point mutation correction and in cDNA knock-in of a codon optimized version the RPK cDNA. We have designed both strategies to maintain the endogenous regulation of RPK once the gene editing has been carried out. First, we designed specific sgRNA targeting NM_000298.5(PKLR):c.359C>T mutation reported as pathogenic in PKD patients, and a single-stranded donor oligonucleotide (ssODN) to correct this mutation. When both sgRNA/Cas9 ribonucleoprotein (RNP) and ssODN were nucleofected together in a heterozygous PKD patient-lymphoblastic cell line (PKD LCL), around 5% of total alleles were correctly edited. In a second strategy, we developed a knock-in gene editing strategy at the genomic starting site of the PKLR gene by combining RNP electroporation and the adeno-associated viral vector (AAV) delivery of the recombination matrix. Specific gRNAs generating up to 60% indels at the RPK starting site were generated. Two different AAV constructs flanked by specific homologous arms were generated to delivery either a TurboGFP expression cassette or a promotor-less therapeutic coRPK. Up to 60% donor integration and stable expression of turbo EGFP and of coRPK driven by PKLR endogenous promoter was obtained in K562 erythroleukemia cells. Similar gene editing efficacies were obtained in human CB-CD34+. Specific integration and stable expression of the transgenes were detected in up to 30% colony forming units (CFUs). Moreover, gene edited cells engrafted efficiently in NSG mice. These results demonstrate the feasibility of editing the PKLR locus in hematopoietic progenitors and hematopoietic stem cells at efficiencies that could be clinically applicable to treat Pyruvate Kinase deficiency. Disclosures Bueren: Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Porteus:CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Segovia:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Summary: Several models of hematopoietic reconstitution after transplant have been proposed with multiple methods of tracking individual clones in both animal models and patients. Lentivirus-mediated gene therapy is the most widely applied strategy for marking individual cells, tracked by either provirus integration site analysis (ISA) or by proviral DNA barcode sequencing (DBS). Two previous studies have applied one of each of these methods to track hematopoiesis in a nonhuman primate model of myeloablative autologous transplantation. The report using ISA described long-term, multi-lineage hematopoietic clones emerging at 1 year after transplantation [Kim S. et al., Cell Stem Cell, 2014]. The report using DBS described only early engraftment up to 9 months after transplant, precluding long term analysis [Wu C. et al., Cell Stem Cell, 2014]. We applied a combination of ISA and DBS methods to follow longitudinal hematopoiesis in a total of six nonhuman primates following autologous transplant in the myeloablative setting, tracking >120,000 individual clones for up to 10 years. We measured hematopoietic lineage reconstitution, as well as spatial and temporal contributions of individual clones within the bone marrow niche and peripheral blood. Here we applied a stringent definition of the hematopoietic stem cell (HSC) phenotype in vivo (i.e. shared clonal identity between short-lived granulocytes and long-lived B cells with multiple time points of detection), to assess HSC contributions after engraftment. Strikingly, we demonstrate sustained contribution of HSC clones occurs as early as 1 month after transplant and that minor clonal waves continuously emerge when the measureable clonal pool is small in size, but not when the initial engrafting clonal pool is robust. Nearly one half of all HSC clones identified over the length of follow-up were detectable in the periphery within a few months after transplant by both ISA and DBS methods. However, we also identify technical differences between these clone tracking methods that can obscure biological interpretation. This study demonstrates an early and robust phase of multi-lineage hematopoietic reconstitution after myeloablative transplantation in the nonhuman primate, suggesting a single subpopulation of CD34+ cells which maintains clonal stability over the lifetime of the recipient in the autologous setting. Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership. Porteus:CRISPR Therapeutics: Consultancy, Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.

Pediatric patients with hematologic malignancies undergoing allogeneic hematopoietic stem cell transplantation (aHSCT) comprise a patient cohort at highest risk of developing disseminated fungal infections. Invasive aspergillosis post-HSCT is associated with unacceptably high mortality in pediatric patients with overall survival of only 15-34%. Hospital construction and excavation further potentiate the risk of fungal related morbidity and mortality in the post-HSCT setting. The current standard of care for anti-fungal prophylaxis for children undergoing aHSCT is the use of triazoles. However, 40% of healthcare associated mold infections in pediatric leukemia patients exposed to hospital construction are historically resistant to voriconazole. Methods to optimize prevention of fungal infections in this high-risk population are critical to improving pediatric aHSCT survival outcomes. We hypothesized that pre-emptive ambisome prophylaxis would be tolerated in the post-HSCT setting and would decrease the incidence of voriconazole resistant healthcare-associated fungal infections and resultant morbidity. We explored the use of intermediate dose daily ambisome (3mg/kg/day) as fungal prophylaxis in 5 patients with hematologic malignancies (2=ALL, 2=AML, 1=Lymphoblastic lymphoma) undergoing allogeneic stem cell transplantation in proximity to construction and excavation. Patient demographics, disease, donor source, conditioning regimen, methotrexate course and transplant related morbidity are shown in table 1. We found that 5 of 5 patients experienced delayed clearance of low dose methotrexate (MTX). This delayed clearance resulted in delayed methotrexate dosing in 3 of 5 patients and a missed methotrexate dose in 1 patient who subsequently developed GVHD and complications from high dose steroids. 4 of 5 patients experienced acute kidney injury (AKI), demonstrated by doubling of the pre-HSCT creatinine value following MTX administration. This identified creatinine elevation is increased as compared to historical controls receiving standard anti-fungal prophylaxis with azole agents. It has been described that AKI delays the clearance of high dose methotrexate. Here, we demonstrate that intermediate doses of daily ambisome following allogeneic HSCT is associated with AKI and delayed clearance of low doses of methotrexate used for GVHD prophylaxis in pediatric patients. Delayed methotrexate clearance was associated with development of morbidity including inadequate GVHD prophylaxis and possible resultant development of GVHD. Combined ambisome and low dose MTX is associated with AKI and delayed MTX clearance and thus is a constrained strategy for prevention of invasive fungal disease in the post-HSCT pediatric setting. The implication of these finding can be extrapolated beyond the construction setting implying cautious use of intermediate dose ambisome prophylaxis in combination with low dose MTX GVHD prophylaxis in pediatric patients post aHSCT. Table 1 Table 1. Disclosures Porteus: CRISPR Therapeutics: Consultancy, Equity Ownership.

Naturally occurring, large deletions in the β-globin locus result in increased fetal hemoglobin (HbF) expression (HPFH, Hereditary Persistence of Fetal Hemoglobin), a condition that mitigates the clinical severity of Sickle Cell Disease (SCD) and β-thalassemia. Here, we integrated BCL11A and GATA1 transcription factor binding site analysis and HPFH mutational data to identify potential HbF silencers in the β-globin locus. Based on this analysis, we designed a CRISPR/Cas9 strategy to disrupt: (i) a 3.5-kb δγ-intergenic region that is specifically deleted in individuals with HPFH. This region contains BCL11A and GATA1 binding sites in adult, HbF-negative primary erythroblasts, thus representing a potential HbF silencer; (ii) a 7.2-kb region, encompassing the 3.5-kb δγ-intergenic element, which is the minimal naturally occurring deletion associated with HPFH ("Corfù" deletion; Chakalova et al., Blood, 2005); (iii) a 13.6-kb genomic region commonly deleted in HPFH, which includes the δ- and β-globin genes and putative intergenic HbF silencers. Targeted deletion or inversion of the 13.6-kb genomic region caused a substantial increase in γ-globin mRNA levels, re-activation of HbF synthesis and a concomitant decrease in HbA expression in adult human erythroid cells (HUDEP-2). Interestingly, deletion of the Corfù region or of the 3.5-kb putative HbF silencer, increased γ-globin gene transcription but failed to produce accumulation of γ-globin mRNA, suggesting a post-transcriptional regulation of γ-globin synthesis in the presence of an intact and active β-globin gene. We then tested re-activation of HbF synthesis in primary adult hematopoietic stem/progenitor cells differentiated towards the erythroid lineage in liquid and clonogenic cultures. Targeting the 13.6-kb genomic region resulted in a high proportion of γ-globin expressing primary erythroblasts, with HbF representing up to 50% of total hemoglobin. Cell morphology, erythroid marker profile, total hemoglobin levels and erythroid maturation were unaffected, consistent with the asymptomatic phenotype of adult HPFH carriers. These data suggest that this region could serve as target for therapeutic genome editing for HbF induction in β-hemoglobinopathies. Overall, this study contributes to the knowledge of the mechanisms underlying fetal to adult Hb switching, and provides clues for a genome editing approach to the treatment of SCD and β-thalassemia. Disclosures Cradick: CRISPR Therapeutics: Employment. Lundberg:CRISPR Therapeutics: Employment, Equity Ownership. Porteus:CRISPR Therapeutics: Consultancy, Equity Ownership. Mavilio:Adverum Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding.

CRISPR/Cas9 systems provide a powerful tool for precision genome editing due to their highly efficient targeting of specific DNA sequences in a genome. We have designed CRISPR single-guide RNAs (sgRNAs) containing the guide and tracr RNAs to selectively edit the human globin locus for potential therapeutic benefit in treating sickle cell anemia and beta-thalassemia. Multiple sgRNAs have been identified and tested across key sites in the human globin locus, and the gene modification frequencies quantified in 293T, K562 and CD34+ hematopoietic progenitor cells. The nuclease cutting efficiency was determined by the rate of insertions, deletions or mutations (indels) from NHEJ-directed repair. The indel rates for these sgRNAs ranged from Disclosures Porteus: CRISPR Therapeutics: Consultancy, Equity Ownership. Cornu:CRISPR Therapeutics: Research Funding. Miccio:CRISPR Therapeutics: Research Funding. Cradick:CRISPR Therapeutics: Employment. Cathomen:CRISPR Therapeutics: Consultancy, Research Funding. Lundberg:CRISPR Therapeutics: Employment. Mavilio:CRISPR Therapeutics: Consultancy, Research Funding.

Chromosomal rearrangements involving the MLL gene occur in both primary and treatment-related leukemias, and are associated with a poor prognosis. While animal models of MLL-AF9 translocations have improved our understanding of the role of MLL oncogenes in leukemia pathogenesis, in this study a more representative approach based on generation of endogenous activated oncogenes is used to more faithfully model the genomic events on human leukemia. This system allows for prospective investigation of the downstream effects of the initiating event on gene expression, epigenetic regulation, stem cell biology, and genome stability in order to understand the key steps critical for the pathogenesis of MLL-rearranged leukemias. We designed a set of TALENs that cut in intron 11 of the MLL gene (MLL-11 TALENs) to facilitate insertion of sequences encoding for AF9 and the fluorescent marker gene coding NeonGreen (knock-in approach). This resulted in MLL-AF9 expression controlled through the endogenous MLL promoter. Co-expression of MLL-11 TALENs with the AF9 knock-in template resulted in AF9 NeonGreen insertion both in K562 cells and in primary human hematopoietic stem cells isolated from umbilical cord blood. This approach showed a knock-in efficiency of ~20% (range 2-40%) in K562 and CD34+ cells based on NeonGreen expression detected by flow cytometry and confocal microscopy. Long-term culture of primary human hematopoietic cells showed increased frequency of knock-in cells suggesting a survival advantage. Remarkably, further enrichment of knock-in cells was promoted by colony-forming assays in semi-solid medium. Subsequent transplantation into NSG mice was performed to assess their leukemogenic potential. Strikingly, leukemia was induced within 8-14 weeks post transplantation. All mice presented with a similar disease profile that included peripheral blood blasts and splenomegaly. Histologic examination confirmed extensive replacement of bone marrow cells and splenic infiltration by leukemic blasts expressing MLL-AF9 detected by RT-PCR. The leukemic blasts showed increased levels of common MLL target genes (HoxA9, Meis1) compared to non-MLL leukemias and comparable levels to known MLL-AF9 cell lines (Mono Mac 6, THP-1). The blast cells displayed a pre-lymphoid phenotype with CD10+/CD19++/CD38++/CD34+ and were negative for mature B cell markers CD20 and IgM as measured by flow cytometry. Our studies establish a novel experimental model to generate MLL leukemia deriving from primary human hematopoietic stem cells expressing the fusion oncogene under control of the endogenous promoter. The model will allow for further prospective study of leukemia initiating and stem cell biology for a genetic subtype of poor prognosis leukemia. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem cell transplantation (HSCT) has been used to cure patients for over four decades, yet the kinetics and clonal dynamics of repopulation by multipotent HSCs remains unresolved. Two sequence-based methods have been used to measure HSC contribution in preclinical models, integration site (IS) analysis and DNA barcoding. Both methods are accomplished by retrovirus tagging of HSCs and each confers different methodological constraints. We hypothesized that IS analysis and DNA barcoding together would provide more robust HSCT reconstitution data if applied to the clinically relevant nonhuman primate autologous HSCT model. We developed a high complexity (~1.2 million), DNA-barcoded LV library encoding the chemotherapy resistance gene MGMT(P140K) and enhanced green fluorescent protein (GFP) to measure hematopoietic reconstitution following total body irradiation (1020 cGy) and autologous HSCT in two pigtailed macaques. In both animals, hematopoietic recovery from TBI was achieved within 1-2 months after HSCT. We observed ~1 and 12% GFP+ peripheral blood white blood cells (PB WBCs) following hematopoietic recovery in these animals, respectively. We performed barcode and IS retrieval from PB WBCs collected early (1 month) after HSCT and observed 1 in 10,000 infused CD34+ cells is capable of trilineage hematopoiesis. In sum, the combination of retrovirus integration site analysis with molecular barcode analysis provides synergistic methods in understanding the kinetics and clonal dynamics of hematopoietic reconstitution following autologous HSCT and revealed a successive pattern of multi-lineage clonal behavior. Disclosures No relevant conflicts of interest to declare.

Introduction Chromosomal rearrangements involving the MLL gene occur in both primary and treatment-related leukemias and convey a poor prognosis. Infants with acute lymphoblastic leukemia (ALL) containing an MLL translocation have a 5-year event free survival of 35% as compared to 69% for infants with germline MLL (Kang et al., Blood 2012). While animal models of MLL-AF9 translocations typically found in adult AML have improved our understanding of the role of MLL translocations in leukemia pathogenesis, modeling the MLL-AF4 translocation commonly found in infant ALL has proven difficult. We hypothesized that recent advances in genome engineering would facilitate induction of specific MLL translocations in primary hematopoietic stem cells to recapitulate the initiating event that leads to infant leukemia. A better understanding of the pathogenesis of the MLL-AF4 translocation will allow for the development of targeted therapies to improve outcome in MLL-rearranged infant leukemia. Methods Transcription activator-like effector nucleases (TALENs) were designed to recognize a patient-specific translocation site within the break point cluster region of the MLL gene and the AF4 gene, respectively. These TALEN pairs were then co-expressed in either K562 cells or primary human CD34+ cells isolated from umbilical cord blood to induce patient-specific double strand breaks. Traditional PCR, nested PCR, and high throughput next generation sequencing were used to detect MLL-AF4 translocations, reciprocal AF4-MLL translocations, as well as other MLL translocations. Clonal analysis was performed to determine the translocation efficiency resulting from two site-specific double strand breaks. Primary cells were maintained in long-term culture to assess for potential survival advantage conferred by the translocations. Finally, primary human CD34+ cells with an induced MLL-AF4translocation were transplanted into NSG mice to assess for leukemic potential. Results We successfully generated TALENs that induce a patient-specific double strand break within the breakpoint cluster regions of the endogenous MLL and AF4 genes. Co-expression of the MLL and AF4 TALENs in K562 cells as well as in primary human hematopoietic stem cells isolated from umbilical cord blood resulted in de novo MLL-AF4 translocations as well as the reciprocal AF4-MLL translocation. The MLL-AF4 translocation efficiency was appreciably higher in K562 cells (10-4) compared to primary hematopoietic stem cells (10-5). Long-term culture of the primary human hematopoietic cells showed an increase in the frequency of the MLL-AF4 translocation-positive cells, suggesting a survival advantage for the cells containing the translocation. These cells were subsequently transplanted into NSG mice to assess their ability to induce leukemia. These studies are currently ongoing. Conclusions Advances in genome engineering have provided the tools necessary to study leukemia pathogenesis in a prospective manner. We have designed an experimental system to recapitulate the process of leukemogenesis. TALENs are effective to induce a specific MLL translocation in primary human hematopoietic stem cells with minimal off target effects, under the control of the endogenous promoter, and in the presence of the reciprocal translocation. This system will allow us to prospectively investigate the downstream effects of this initiating event on gene expression, epigenetic regulation, and genome stability in order to understand the key steps critical for the pathogenesis of MLL-rearranged leukemias. Disclosures: No relevant conflicts of interest to declare.

Introduction Genome-wide association studies (GWAS) have ascertained numerous trait-associated common genetic variants localized to regulatory DNA. The hypothesis that regulatory variation accounts for substantial heritability has undergone scarce experimental evaluation. Common variation at BCL11A is estimated to explain ∼15% of the trait variance in fetal hemoglobin (HbF) level but the functional variants remain unknown. Materials and Methods We use chromatin immunoprecipitation (ChIP), DNase I sensitivity and chromosome conformation capture to evaluate the BCL11A locus in mouse and human primary erythroblasts. We extensively genotype 1,263 samples from the Collaborative Study of Sickle Cell Disease within three HbF-associated erythroid DNase I hypersensitive sites (DHSs) at BCL11A. We pyrosequence heterozygous erythroblasts to assess allele-specific transcription factor binding and gene expression. We conduct transgenic analysis by mouse zygotic microinjection and genome editing with transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) RNA-guided nucleases. Results Common genetic variation at BCL11A associated with HbF level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping this putative regulatory DNA uncovers a motif-disrupting common variant associated with reduced GATA1 and TAL1 transcription factor binding, modestly diminished BCL11A expression and elevated HbF. This variant, rs1427407, accounts for the HbF association of the previously reported sentinel SNPs. The composite element functions in vivo as a developmental stage-specific lineage-restricted enhancer. Genome editing reveals that the enhancer is required in erythroid but dispensable in B-lymphoid cells for expression of BCL11A. We demonstrate species-specific functional components of the composite enhancer in mouse as compared to human erythroid precursor cells. The mouse sequences homologous to the human DHS sufficient to drive reporter activity are dispensable from the mouse composite element, whereas the adjacent DHS, whose human homolog does not direct reporter activity, is absolutely required for BCL11A expression. Conclusions We describe a comprehensive and widely applicable approach, including chromatin mapping followed by fine-mapping, allele-specific ChIP and gene expression studies, and functional analyses, to reveal causal variants and critical elements. We assert that functional validation of regulatory DNA ought to include perturbation of the endogenous genomic context by genome editing and not solely rely on in vitro or ectopic surrogate assays. These results validate the hypothesis that common variation modulates cell type-specific regulatory elements, and reveal that although functional variants themselves may be of modest impact, their harboring elements may be critical for appropriate gene expression. We speculate that species-level functional differences in components of the composite enhancer might partially account for differences in timing of globin gene expression among animals. We suggest that the GWAS-marked BCL11A enhancer represents a highly attractive target for therapeutic genome editing for the major b-hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.

Abstract 1398 Introduction: Acute myeloid leukemia (AML) is a heterogeneous malignancy that continues to have a significant relapse rate in both adult and pediatric patients. This heterogeneity manifests itself between patients because different patients will have different gene mutations causing their AML (inter-patient heterogeneity). It also manifests as intra-patient heterogeneity as within a single patient there are different sub-clones that contribute differentially to primary disease and relapse. For example, relapse after chemotherapy can occur from a genetically distinct sub-clone that presumably was relatively resistant to the administered chemotherapy. The dynamics of clonal relapse and the contribution of intra-patient functional heterogeneity (of either genetic or epigenetic origin), however, remains poorly understood. We have developed an assay using a molecular barcode system and next generation sequencing to simultaneously track the fate of ∼14,000 different clones either in vitro or in vivo (Figure 1). Individual clones can therefore be tracked over time even in complex populations. We validated this assay on a human AML cell line, THP1, over multiple population doublings, describe how the heterogeneity can be monitored and quantified over time and describe that even in this cell line the population continues to undergo dynamic clonal changes. METHODS: A lentiviral barcode library consisting of ∼14,000 unique 20 basepair sequences (the molecular barcode) was transduced into THP1 cells at a multiplicity of infection of 0.05 (to assure that each cell only acquired a single barcode). Barcoded cells were purified by selection for GFP by flow cytometry. Cells were maintained in RPMI at a concentration of 2×106 cells/ml in triplicates and passaged every 2∼3 days. The percentage of GFP expression was assessed every 30 population doublings (PD). Cells were passaged to a maximum of 90 population doublings. Genomic DNA was extracted every 10 population doublings (PD), barcodes were amplified by PCR using primers bracketing the barcode region to produce a 250 bp product, and gel purified 250 bp products were submitted to Illumina™ high throughput sequencing to quantify the abundance of each barcode in the population. Barcodes were clustered allowing for a minimum quality score of 30 and a maximum of 1 mismatch. Clusters were ordered and plotted by frequency of occurrence from highest to lowest. The frequency of barcode clusters at specific PD was compared between the triplicates. RESULTS: The population doublings and percentage of GFP expression of barcoded THP1 cells was nearly identical in the triplicate cell passage of up to 90 population doublings (Figure 2. THP1 bc1 = barcoded THP1 tissue passage 1 of triplicate, THP1 bc2 = barcoded THP1 tissue passage 2 of triplicate, THP1 bc3 = barcoded THP1 tissue passage 3 of triplicate). A high fraction of these barcoded cells (> 90%) expressed GFP consistently throughout the 90 population doublings. On the other hand the heterogeneity, represented by clusters of barcodes in a single cell passage at PD 30, 60 and 90, varied between PD as shown in the stacked histogram (Figure 3). PD 30 was the reference PD and was sorted from highest cluster frequency to lowest; all other PDs were compared to PD 30. CONCLUSIONS: Subpopulations of cells are dynamic even in established cell lines, including the THP1 human AML cell line. Fluctuations in barcode frequencies were observed between PD within a sequential cell passage. A high degree of heterogeneity was therefore maintained and thus represents the highly dynamic nature of this AML cell line. This barcode assay can be a powerful tool to assess how population heterogeneity can be affected by environmental pressures such as chemotherapy. Our future effort is directed at challenging barcoded THP1 cells with cytarabine, a foundation of AML therapy, to determine how chemotherapy affects the functional heterogeneity of a dynamic heterogeneous population. In addition, we are also assessing how heterogeneity is influenced in vivo by first transplanting barcoded THP1 cells into NSG mice and then challenging the transplanted mice with chemotherapy. Disclosures: Hsu: Child Health Research Institute and the Stanford CTSA (grant no. UL1 RR025744): Research Funding; Rosa Wann and Marjorie Shannon Fellowship: Research Funding. Porteus:Alex's Lemonade Stand Foundation: Research Funding.

Abstract 828 Genome-wide association studies (GWAS) have identified common genetic variation within a 14-kb segment of intron-2 of the BCL11A gene that is associated with the level of fetal hemoglobin (HbF) and the clinical severity of the β-globin disorders sickle cell disease and β-thalassemia. Functional studies have validated BCL11A as a major repressor of HbF. Here, we explore the consequences of genetic variation on BCL11A expression. The HbF-associated variants overlap BCL11A sequences that possess an evolutionarily conserved enhancer chromatin signature, including the presence of H3K4me1 and H3K27ac and the absence of H3K4me3 histone modifications. Moreover, these sequences show a unique pattern of DNase I hypersensitivity in erythroid precursors compared to other BCL11A-expressing lineages including fetal brain and B-lymphocytes. Occupancy of the major erythroid transcription factors GATA1 and TAL1 is observed at these DNase I hypersensitive sites (DHS). Chromosome conformation capture studies demonstrate that these DHSs form a long-range interaction with the BCL11A promoter. To address the function of the putative regulatory element, we generated transgenic mice with the 12-kb intronic region attached to a minimal promoter and reporter gene. The element enhanced reporter gene expression in an erythroid-lineage restricted, definitive erythroid stage-specific manner. The minimal enhancer activity was mapped to the +58-kb DHS. Similar enhancer activity was observed in primary human erythroid precursors. To address the possible requirement of this element for BCL11A expression, we deleted the corresponding region in adult-stage murine erythroleukemia cells with TALEN-mediated gene targeting. Expression of BCL11A was dramatically reduced and embryonic globins Hbby and Hbb-bh1 were markedly derepressed with concomitant reduction of adult globin Hbb-b1. Analysis of heterozygous primary human erythroid precursors revealed that low-HbF haplotype alleles are preferentially occupied by GATA1 and TAL1 at sequences surrounding rs1427407. A composite half-E-box–GATA motif is disrupted at this SNP in the high-HbF allele. Furthermore, the low-HbF haplotype allele of BCL11A is expressed at a 1.7-fold higher level relative to the high-HbF allele. These data demonstrate that HbF-associated genetic variation in BCL11A modulates the function of a critical erythroid enhancer of BCL11A, and consequently expression of BCL11A itself. The quantitative level of BCL11A in turn regulates the relative expression of the stage-specific globin genes. The essential erythroid enhancer of the BCL11A gene might itself serve as a target for genetic manipulation as a novel approach to the human β-globin disorders. Disclosures: Porteus: Alex's Lemonade Stand Foundation: Research Funding.

Abstract 1410 Leukemias are generally clonal in origin, but develop significant heterogeneity as they expand and acquire genetic and epigenetic alterations. Detecting this heterogeneity and tracking the dynamics and evolution of clones within a population are important for our understanding of these cancers and the phenomena of drug resistance and relapse. Attempts to track the contributions of individual cells to the clonality of large populations, however, have been constrained by limitations in sensitivity and complexity. We have created an efficient and high throughput method to overcome these limitations by harnessing the power of viral marking and next generation sequencing technology, allowing us to track the clonal contributions of many thousands of cells with minimal perturbations to the population as a whole. K562s are a common patient-derived CML line featuring rapidly proliferating, highly aneuploid cells. We stably and heritably marked individual cells via the lentiviral-mediated insertion of one of ∼14,000 random, non-coding, 20 basepair DNA “barcodes.” Using Illumina next-generation sequencing we determined the relative frequency of every barcode, thus directly measuring the contribution of each clone to the overall population over 90 population doublings. We anticipated that the the K562 cell line, while very highly aneuploid, would have stable clonal dynamics because it has been grown in culture for years, thus allowing potentially dominant clones to reach equilibrium. We found, however, that K562 cells continue to undergo dramatic changes in clonal representation. As expected, in analysis of our clonally marked population, there was a broad distribution of clones. After culturing under ideal conditions for 90 population doublings, we found that a single clone now represented 9% of the population and that less than 100 clones now represented more than 50% of the population, a massive clonal skewing after just a brief period of time. Cell lines are often assumed to be a homogenous group of identical clones, a seemingly reasonable assumption considering the years of passaging/subculturing. However, our results show that ongoing genomic and/or epigenomic instability leads to proliferative instability, resulting in unexpected clonal dynamics and rapid clonal evolution. These results have profound implications for the experimental use of cell lines, as well as broad applications for clonal analysis of leukemias, other malignancies, and stem cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 1071 Zinc finger nucleases (ZFNs) are custom-designed DNA binding proteins that produce DNA double-strand breaks (DSBs) at predetermined genomic sites, stimulating homology-directed repair in the presence of donor template by many orders of magnitude over the spontaneous rate. The ability to target specific genes with ZFN technology opens therapeutic opportunities for gene correction and selective gene silencing. Sickle cell anemia (SCA) is an ideal disease target because correction of the single gene β-globin mutation in patient-derived, autologous hematopoietic stem progenitor cells (HSPCs) promises to be curative. One of the barriers to ZFN-based gene correction is the lack of a nonviral delivery system that achieves bulk transport of the nucleases to hard-to-transfect target cells, such as embryonic and HSPCs. To address this challenge, we set out to develop a delivery platform that is (i) gentle to the cell, (ii) provides tunable delivery rates, and (iii) achieves improved spatio-temporal control of the nucleases. To reconcile these goals, we have explored a method for direct delivery of ZFNs as proteins by receptor-mediated endocytosis. We selected the transferrin receptor pathway as our lead candidate on the rationale that all nucleated cells, including HSPCs, must import elemental iron to remain viable under ex vivo culture conditions. To test the feasibility of this strategy, this initial work used a ZFN pair targeted against a model GFP transgene. We optimized expression by pilot scale fermentation in an Escherichia coli host-vector system and purification to homogeneity by serial chromatography. We conjugated the purified ZFNs to the iron carrier protein, transferrin (tf), using SPDP, an amine and sulfhydryl reactive heterobifunctional crosslinker. The resulting disulfide linkage is designed to undergo scission (“self-immolation”) upon entry into the intracellular reducing environment. In vitro DNA cleavage assays and surface plasmon resonance binding assays demonstrated that ZFNs remained competent for target sequence cleavage following conjugation, with only mild to quantitative impairment of activity. To analyze delivery in biological systems, we measured time- and dose-dependence of tf-mediated ZFN uptake in human osteosarcoma (U2OS 2–6–3) cells. ZFNs in DAPI stained cell nuclei were detected by indirect immunofluorescence and signal intensity was measured in projections of deconvolved depth coded z-stacks. Nuclear uptake of tf-ZFN protein occurred in >95% of cells, was dose-dependent and linear with time in the lower dose ranges, and reached saturation as early as 60 min. Importantly, maximal nuclear uptake was indistinguishable from ZFN plasmid treated cells. These results indicate that endocytic delivery of ZFNs readily traverses the cellular membrane, overcomes the potential hurdle of endosomal trapping, and targets the nucleus with high efficiency. To demonstrate gene targeting activity, we used the U2OS 2–6–3 cell assay which bears a tandem transgene array at a single locus that is cleavable by our GFP ZFNs. Cells were transfected with lacI-ECFP to mark the target locus, incubated with tf-ZFNs, fixed, and stained for 53BP1, a signaling protein that marks DSBs. Recruitment of 53BP1 to the target locus was observed in 13% (18/135) of tf-ZFN treated cells, whereas no recruitment (0/152) was observed in untreated cells. These findings demonstrate that tf-conjugated ZFNs retain cleavage activity after nuclear uptake in a significant percentage of cells. To determine whether the tf-ZFNs are capable of stimulating gene correction, we transfected primary mouse adult fibroblasts carrying a mutant GFP transgene with donor template, incubated with tf-ZFNs, and evaluated cells at 72 h for gene correction as evidenced by GFP expression. Flow cytometry revealed a gene correction rate of 1–2%, identical to ZFN plasmid transfected cells, demonstrating that the technology of shuttling ZFN proteins to the cell interior via the tf-receptor pathway can deliver bioactive ZFNs to the nuclear compartment, target specific gene sequences, and induce homology-directed repair in the presence of donor DNA. We are currently testing these methods in hematopoietic stem cells, with the ultimate goal of correcting the sickle globin allele. Toward this end, we plan to adapt these approaches for high-throughput transfer of ZFN proteins directly to the hematopoietic stem progenitor cell. Disclosures: No relevant conflicts of interest to declare.