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β-Thalassemia pathology is due not only to loss of β-globin (HBB), but also to erythrotoxic accumulation and aggregation of the β-globin-binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in β-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize β-globin:α-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing β-thalassemia.

Reticular Dysgenesis is a particularly grave form of severe combined immunodeficiency (SCID) because it affects the adaptive and innate immune system. Patients suffer from congenital neutropenia, defective lymphocyte development and sensorineural hearing loss. The disease is caused by biallelic loss of function mutations in the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) as substrate for adenosine triphosphate (ATP) synthesis. Accordingly, declining OXPHOS metabolism has been postulated as the driver of disease pathology. The mechanistic basis for the failure of myelopoiesis and lymphopoiesis in Reticular Dysgenesis, however, remains incompletely understood. We have used single cell RNA-sequencing of bone marrow cells from two Reticular Dysgenesis patients to gain insight into the disease mechanism. Gene set enrichment analysis of differentially expressed genes in different subsets of myeloid and lymphoid progenitor cells implicated processes involved in RNA catabolism and ribonucleoprotein synthesis in the pathogenesis of Reticular Dysgenesis. To investigate these findings and precisely mimic the failure of human myelopoiesis in culture, we developed a cell-traceable disease model for Reticular Dysgenesis based on CRISPR-mediated disruption of the AK2 gene in primary human hematopoietic stem cells. In this model, we have shown that AK2-deficienct myeloid progenitor cells not only have compromised mitochondrial energy metabolism and increased AMP levels, but also NAD+ and aspartate depletion, metabolites that rely on TCA-cycle activity for regeneration and synthesis. Furthermore, AK2-deficient cells exhibited strikingly increased levels in the purine nucleotide precursor inosine monophosphate (IMP), decreased cellular RNA content and ribosome subunit expression, reduced protein synthesis and a profoundly hypo-proliferative phenotype. Although NAD+ and aspartate are critical substrates of purine synthesis, AK2-deficient cells did not exhibit purine auxotrophy. Instead, our studies revealed that the rise in IMP levels stemmed from increased AMP deamination. Pharmacologic inhibition of AMP deaminase normalized IMP levels in AK2-deficient cells, but further aggravated the Reticular Dysgenesis phenotype, suggesting AMP catabolism to IMP represents a metabolic adaptation to mitigate AMP-mediated toxicity. This study is the first to identify globally curtailed mitochondrial metabolism resulting in NAD+ and aspartate deficiency and disordered purine metabolism as the two primary cellular consequences of AK2-deficiency. Our data suggests that AMP accumulation and its detrimental effects on ribonucleotide synthesis capacity are the dominant driver of cellular pathology causing failure of myelopoiesis in Reticular Dysgenesis.

Sickle cell disease (SCD) is the most common monogenic serious disease with 300,000 births annually worldwide. SCD is autosomal recessive from a single point mutation in codon six of the β-globin gene (HBB) resulting in sickle hemoglobin. Ex vivo β-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) might be an ideal treatment of SCD. We previously developed an HBB gene targeting strategy that utilizes high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce rAAV6-mediated gene correction of the SCD-causing mutation in HSPCs. Here we present foundational translational data that demonstrate the pre-clinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34+ cells from healthy and SCD patient donors (Drug Product-gcHBB-SCD). Notably, we achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing and long-term engraftment in immunodeficient NSG mice, with multi-lineage allele gene correction frequencies of 20% in multiple hematopoietic organs. The long-term safety tumorigenicity/toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity or tumorigenicity from the engrafted gcHBB-SCD Drug Product. Altogether, this preclinical data supports the safety, efficacy, and reproducibility of a gene correction strategy for initiation of a Phase I/II clinical trial for SCD patients.

Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the β-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 ± 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16–19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.

Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell (PKLR) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cell (HSPC) transplant, associated with a significant morbidity and mortality, especially relevant in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR-Cas9 system and donor recombinant adeno-associated vector (rAAV) delivery to build an efficient, safe, and clinically applicable system to knock in therapeutic sequences at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient mice. Erythroid cells derived from edited PKD-HSPCs recovered normal ATP levels, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene-editing strategy may represent a lifelong therapy to correct RPK functionality in RBCs for PKD patients., Graphical abstract, Combination of CRISPR-Cas9 ribonucleoprotein electroporation and donor AGAVE transduction in human hematopoietic stem and progenitor cells from pyruvate kinase deficiency patients allows efficient gene editing with restoration of erythrocyte energetic balance. Maintenance of stem cell properties without detectable off-targets indicates clinical applicability of gene editing for this inherited hemolytic anemia.

CRISPR/Cas9-mediated beta-globin (HBB) gene correction of sickle cell disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated previously. Here, we use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observe a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo red blood cell (RBC) features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research., CRISPR mediated gene correction of sickle cell disease (SCD) in patient-derived hematopoietic stem cells is a promising avenue for therapy. Here the authors use a humanized SCD mouse model to study gene editing in the context of autologous transplantation.

β-thalassemia pathology is not only due to loss of β-globin (HBB), but also erythrotoxic accumulation and aggregation of the β-globin binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in β-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize β-globin:α-globin mRNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in mice, establishing proof-of-concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing β-thalassemia.

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may usher in a new class of medicines to treat genetic diseases of the blood and immune system. With state-of-the-art methodologies, it is now possible to correct disease-causing mutations at high frequencies in HSPCs by combining ribonucleoprotein (RNP) delivery of Cas9 and chemically modified sgRNAs with homologous DNA donors via recombinant adeno-associated viral vector serotype six (AAV6). However, because of the precise nucleotide-resolution nature of gene correction, these current approaches do not allow for clonal tracking of gene targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a novel methodology that utilizes barcoded AAV6 donor template libraries, carrying either in-frame silent mutations or semi-randomized nucleotide sequences outside the coding region, to track the in vivo lineage contribution of gene targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ∼20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in immunodeficient mice. We anticipate that this methodology has the potential to be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings., Genetic barcoding has been used to track clonal dynamics of cells. Here, the authors develop a Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), to barcode repaired alleles by introducing silent mutations or outside of coding regions, to show clonal complexity of edited CD34 + cells following engraftment.

Genome-editing technologies are currently being translated to the clinic. However, cellular effects of the editing machinery have yet to be fully elucidated. Here, we performed global microarray-based gene expression measurements on human CD34(+) hematopoietic stem and progenitor cells that underwent editing. We probed effects of the entire editing process as well as each component individually, including electroporation, Cas9 (mRNA or protein) with chemically modified sgRNA, and AAV6 transduction. We identified differentially expressed genes relative to control treatments, which displayed enrichment for particular biological processes. All editing machinery components elicited immune, stress, and apoptotic responses. Cas9 mRNA invoked the greatest amount of transcriptional change, eliciting a distinct viral response and global transcriptional downregulation, particularly of metabolic and cell cycle processes. Electroporation also induced significant transcriptional change, with notable downregulation of metabolic processes. Surprisingly, AAV6 evoked no detectable viral response. We also found Cas9/sgRNA ribonucleoprotein treatment to be well tolerated, in spite of eliciting a DNA damage signature. Overall, this data establishes a benchmark for cellular tolerance of CRISPR/Cas9-AAV6-based genome editing, ensuring that the clinical protocol is as safe and efficient as possible.

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 .

The hematopoietic system is responsible for transporting oxygen and nutrients, fighting infections, and repairing tissue damage. Hematopoietic system dysfunction therefore causes a range of serious health consequences. Lifelong hematopoiesis is maintained by repopulating multipotent hematopoietic stem cells (HSCs) that replenish shorter-lived, mature blood cell types. A prokaryotic mechanism of immunity, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system, has been recently "repurposed" to mutate mammalian genomes efficiently and in a sequence-specific manner. The application of this genome-editing technology to hematology has afforded new approaches for functional genomics and even the prospect of "correcting" dysfunctional HSCs in the treatment of serious genetic hematological diseases. In this Perspective, we provide an overview of three recent CRISPR/Cas9 methods in hematology: gene disruption, gene targeting, and saturating mutagenesis. We also summarize the technical considerations and advice provided during the May 2017 International Society of Experimental Hematology New Investigator Committee webinar on the same topic.

Summary Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

The CRISPR–Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR–Cas9 system to therapeutically correct human genetic diseases1–6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR–Cas9 system moves toward clinical trials. Cas9-specific antibodies and reactive T cells are found in the majority of healthy adult human serum samples analyzed. Such preexisting adaptive immunity should be taken into consideration as the CRISPR–Cas9 system moves toward clinical trials.

The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.

Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene that lead to a reduction of the erythroid pyruvate kinase (RPK) protein activity, which causes an energetic imbalance in PKD erythroid cells. This disease is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. In selected cases, allogeneic Hematopoietic Stem Cell Transplantation has been shown to correct the disorder. Therefore, autologous HSCT of genetically corrected cells will offer a durable and curative therapeutic option. In fact, a global Phase I clinical trial (clinicaltrials.gov #NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy with severe PKD. Looking for a guided integration of the exogenous therapeutic DNA sequences, herein we conducted a gene editing approach to correct PKD in human Hematopoietic Stem Cells (HSCs). We developed a knock-in approach to insert either a TurboGFP expression cassette or a promotor-less therapeutic codon optimized RPK cDNA (coRPK) at the genomic starting site of the PKLR gene, a gene editing strategy that will correct most of the mutations present in PKD patients. This gene editing approach combines RNP nucleofection and adeno-associated viral vector (AAV6) mediated delivery of homologous donors. In order to assess the safety of the proposed gene editing approach, we performed GUIDE-Seq and rhAmpSeqTM analyses of different single guide RNAs targeting the PKLR starting site. We found PKLR sgRNAs that showed a high targeting efficiency, up to 40% targeted hematopoietic progenitors (in vitro semisolid colony forming units) and a safety profile, with no off-targets detected above threshold values (0.1%) and in the absence of cellular toxicity, when applied to healthy cord blood CD34+ (CB-CD34+) cells. These gene-edited CB-CD34+ cells engrafted efficiently in both primary and secondary immunodeficient NSG recipient mice, demonstrating the gene editing in long-term hematopoietic repopulating HSCs. Furthermore, we evaluated the therapeutic potential of this gene editing strategy to restore the energetic imbalance in erythroid cells derived from PKD patients. CD34+ cells from 4 different PKD patients, were purified, nucleofected with PKLR sgRNA and transduced with AAV-coRPK donor. In vitro differentiated erythroid cells derived from edited PKD CD34+ expressed coRPK mRNA and restored their energetic defect up to normal values obtained in in vitro differentiated erythroid cells from healthy donor cells. Moreover, gene edited mobilized Peripheral Blood CD34+ (mPB-CD34+) cells from a PKD patient engrafted efficiently in immunodeficient NBSGW mice, having human cells that showed the specific integration of coRPK donor 2 months post-transplant. Overall, these results demonstrate the feasibility and safety of PKLR gene editing in human HSPCs and, therefore, its potential clinical application for the treatment of PKD patients. Disclosures Dever: Integral Medicines: Current Employment. Turk:Integrated DNA Technologies, Inc. (IDT): Current Employment, Current equity holder in publicly-traded company. Bianchi:Agios Pharmaceuticals: Other: Scientific Advisor. Behlke:Integrated DNA Technologies, Inc. (IDT): Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

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.

With the ability to reconstitute the entire hematopoietic system, hematopoietic stem cells (HSCs) are an attractive target for a range of disease therapies. CRISPR/Cas9-mediated beta-globin (HBB) gene correction of Sickle Cell Disease (SCD) patient-derived HSCs in combination with autologous transplantation represents an exciting new paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated to date. Here, we have applied a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation using a novel ex vivo HSC culture system (Wilkinson et al., Nature 2019). We have discovered that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation back into SCD mice. There was a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo RBC features, but even low levels of chimerism resulted in significant HgbA levels. Our results provide a useful framework for evaluating Cas9-based HSC gene therapies and support the clinical investigation of Cas9-AAV6 technologies.

Precise and efficient manipulation of genes is crucial for understanding the molecular mechanisms that govern human hematopoiesis and for developing novel therapies for diseases of the blood and immune system. Current methods do not enable precise engineering of complex genotypes that can be easily tracked in a mixed population of cells. We describe a method to multiplex homologous recombination (HR) in human hematopoietic stem and progenitor cells and primary human T cells by combining rAAV6 donor delivery and the CRISPR/Cas9 system delivered as ribonucleoproteins (RNPs). In addition, the use of reporter genes allows FACS-purification and tracking of cells that have had multiple alleles or loci modified by HR. We believe this method will enable broad applications not only to the study of human hematopoietic gene function and networks, but also to perform sophisticated synthetic biology to develop innovative engineered stem cell-based therapeutics.

Combination of genome editing and human pluripotent stem cells (hPSCs) offers a platform for in vitro disease modeling, drug discovery and personalized stem cell therapeutics. However, incorporation of large modifications using CRISPR/Cas9-based genome editing in hPSCs typically requires the use of selection markers due to low editing efficiencies. Here we report a novel editing technology in hPSCs using Cas9 protein complexed with chemically modified single guide RNA (sgRNA) and recombinant AAV6 (rAAV6) vectors for donor delivery without marker selection. With these components, we demonstrate targeted integration of a 2.2 kb DNA expression cassette in hPSCs at frequencies up to 94% and 67% at the HBB and MYD88 loci, respectively. We used this protocol to correct the homozygous sickle cell disease (SCD) mutation in an iPSC line derived from a SCD patient with a frequency of 63%. This Cas9/AAV6 system allows for both the integration of large gene cassettes and the creation of single nucleotide changes in hPSCs at high frequencies, eliminating the need for multiple editing steps and marker selection, thus increasing the potential of editing human pluripotent cells for both research and translational applications.

The CRISPR-Cas9 system has proven to be a powerful tool for genome editing, allowing for the precise modification of specific DNA sequences within a cell. Many efforts are currently underway to use the CRISPR-Cas9 system for the therapeutic correction of human genetic diseases. The most widely used homologs of the Cas9 protein are derived from the bacteriaStaphylococcus aureus(S. aureus) andStreptococcus pyogenes(S. pyogenes). Based on the fact that these two bacterial species cause infections in the human population at high frequencies, we looked for the presence of pre-existing adaptive immune responses to their respective Cas9 homologs, SaCas9 (S. aureushomolog of Cas9) and SpCas9 (S. pyogeneshomolog of Cas9). To determine the presence of anti-Cas9 antibodies, we probed for the two homologs using human serum and were able to detect antibodies against both, with 79% of donors staining against SaCas9 and 65% of donors staining against SpCas9. Upon investigating the presence of antigen-specific T-cells against the two homologs in human peripheral blood, we found anti-SaCas9 T-cells in 46% of donors. Upon isolating, expanding, and conducting antigen re-stimulation experiments on several of these donors’ anti-SaCas9 T-cells, we observed an SaCas9-specific response confirming that these T-cells were antigen-specific. We were unable to detect antigen-specific T-cells against SpCas9, although the sensitivity of the assay precludes us from concluding that such T-cells do not exist. Together, this data demonstrates that there are pre-existing humoral and cell-mediated adaptive immune responses to Cas9 in humans, a factor which must be taken into account as the CRISPR-Cas9 system moves forward into clinical trials.

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The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic-helix-loop-helix/PER-ARNT-SIM(PAS) transcription factor superfamily that also mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence suggests that AhR influences the development of many tissues, including the central nervous system. Our previous studies suggest that sustained AhR activation by TCDD and/or AhR deletion disrupts cerebellar granule neuron precursor (GNP) development. In the current study, to determine whether endogenous AhR controls GNP development in a cell-autonomous manner, we created a GNP-specific AhR deletion mouse, AhR(fx/fx) /Math1(CRE/+) (AhR CKO). Selective AhR deletion in GNPs produced abnormalities in proliferation and differentiation. Specifically, fewer GNPs were engaged in S-phase, as demonstrated by ∼25% reductions in thymidine (in vitro) and Bromodeoxyuridine (in vivo) incorporation. Furthermore, total granule neuron numbers in the internal granule layer at PND21 and PND60 were diminished in AhR conditional knockout (CKO) mice compared with controls. Conversely, differentiation was enhanced, including ∼40% increase in neurite outgrowth and 50% increase in GABARα6 receptor expression in deletion mutants. Our results suggest that AhR activity plays a role in regulating granule neuron number and differentiation, possibly by coordinating this GNP developmental transition. These studies provide novel insights for understanding the normal roles of AhR signaling during cerebellar granule cell neurogenesis and may have important implications for the effects of environmental factors in cerebellar dysgenesis.

Since the discovery two decades ago that programmable endonucleases can be engineered to modify human cells at single nucleotide resolution, the concept of genome editing was born. Now these technologies are being applied to therapeutically relevant cell types, including hematopoietic stem cells (HSC), which possess the power to repopulate an entire blood and immune system. The purpose of this review is to discuss the changing landscape of genome editing in hematopoietic stem cells (GE-HSC) from the discovery stage to the preclinical stage, with the imminent goal of clinical translation for the treatment of serious genetic diseases of the blood and immune system.With the discovery that the RNA-programmable (sgRNA) clustered regularly interspace short palindromic repeats (CRISPR)-Cas9 nuclease (Cas9/sgRNA) systems can be easily used to precisely modify the human genome in 2012, a genome-editing revolution of hematopoietic stem cells (HSC) has bloomed. We have observed that over the last 2 years, academic institutions and small biotech companies are developing HSC-based Cas9/sgRNA genome-editing curative strategies to treat monogenic disorders, including β-hemoglobinopathies and primary immunodeficiencies. We will focus on recent publications (within the past 2 years) that employ different genome-editing strategies to 'hijack' the cell's endogenous double-strand repair pathways to confer a disease-specific therapeutic advantage.The number of genome-editing strategies in HSCs that could offer therapeutic potential for diseases of the blood and immune system have dramatically risen over the past 2 years. The HSC-based genome-editing field is primed to enter clinical trials in the subsequent years. We will summarize the major advancements for the development of novel autologous GE-HSC cell and gene therapy strategies for hematopoietic diseases that are candidates for curative allogeneic bone marrow transplantation.

Summary Human neural stem cells (NSCs) offer therapeutic potential for neurodegenerative diseases, such as inherited monogenic nervous system disorders, and neural injuries. Gene editing in NSCs (GE-NSCs) could enhance their therapeutic potential. We show that NSCs are amenable to gene targeting at multiple loci using Cas9 mRNA with synthetic chemically modified guide RNAs along with DNA donor templates. Transplantation of GE-NSC into oligodendrocyte mutant shiverer-immunodeficient mice showed that GE-NSCs migrate and differentiate into astrocytes, neurons, and myelin-producing oligodendrocytes, highlighting the fact that GE-NSCs retain their NSC characteristics of self-renewal and site-specific global migration and differentiation. To show the therapeutic potential of GE-NSCs, we generated GALC lysosomal enzyme overexpressing GE-NSCs that are able to cross-correct GALC enzyme activity through the mannose-6-phosphate receptor pathway. These GE-NSCs have the potential to be an investigational cell and gene therapy for a range of neurodegenerative disorders and injuries of the central nervous system, including lysosomal storage disorders., Graphical Abstract, Highlights • Human neural stem cells are amenable to CRISPR-Cas9-mediated gene targeting • Truncated CD19 surface marker allows for enrichment of gene-targeted NSCs to >90% • Gene-targeted NSCs engraft, migrate, and differentiate in immunodeficient mice • GALC-engineered overexpressing NSCs cross-correct Krabbe disease fibroblasts in vitro, Molecular Biology; Neuroscience; Bioengineering; Biotechnology; Cell Biology; Biological Sciences Research Methodologies

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.

Thirty-six participants were tested on inattentional blindness (IB), inattentional insensitivity for tactile stimuli, and inattentional insensitivity for olfactory stimuli. Inattentional insensitivity (IIS) is the inability to detect the sensation of a salient stimulus while performing a task within a congruent sensory modality. The Invisible Gorilla video (Simons, 2003) was used to test IB. Half of the participants were tasked while engaged in each sensory condition, and half of the participants were not tasked. Chi square analyses showed that tasked participants displayed more IB than nontasked participants. In addition, tasked participants in the tactile condition displayed more IIS for a salient tactile stimulus than participants who were not tasked. No differences were found between groups for IIS in the olfactory condition. High working memory (WM) participants showed better performance than low WM participants in the tactile task. High WM participants also showed better performance during the visual condition (i.e., higher accuracy for basketball counts). Finally, results suggest a possible relationship between IIS with performance across different sensory modalities.

Eighty-two participants were categorized into one of three groups: participants with inattentional blindness (IB), participants without IB, and participants with IB, but were told about their failure to notice a salient, although irrelevant stimulus (the Show group). The Invisible Gorilla video (Simons, 2003) was used to determine IB. The effects of pointing out IB failures were tested for subsequent tasks. Performance and situation awareness (SA) of the Show group in a subsequent task of watching driving videos was compared to the IB group and the group who did not display IB. No differences in performance were found between any of the groups. The IB group was slower to answer irrelevant questions about the driving environment than participants who did not display IB. The IB group and the Show group answered irrelevant questions slower than they answered relevant questions. However, the time to answer relevant SA questions and accuracy of answering SA questions about the driving environment did not differ between the three groups. Thus, it appears that pointing out failures of an irrelevant stimulus has no effect for improving performance or SA of relevant information in subsequent tasks. Participants were further categorized as high working memory or low working memory (WM). IB participants with low WM were slower answering SA questions than IB participants with high WM.

The aryl hydrocarbon receptor (AhR), a ligand-activated member of the basic helix-loop-helix (bHLH)/PER-ARNT-SIM (PAS) transcription superfamily, is known to regulate the toxicity of polyaromatic halogenated hydrocarbon environmental chemicals, most notably dioxin. However, the AhR has also been implicated in multiple stages of tumorigenesis. Medulloblastoma (MB), a primary cerebellar brain tumor arising in infants and children, is thought to originate from abnormally proliferating cerebellar granule neuron precursors (GNPs). GNPs express high levels of the AhR in the external germinal layer of the developing cerebellum. Moreover, our laboratory has previously reported that either abnormal activation or deletion of the AhR leads to dysregulation of GNP cell cycle activity and maturation. These observations led to the hypothesis that the AhR promotes the growth of MB. Therefore, this study evaluated whether the AhR serves a pro-proliferative role in an immortalized MB tumor cell line (DAOY). We produced a stable AhR knockdown DAOY cell line [AhR short hairpin RNA (shRNA)], which exhibited a 70% reduction in AhR protein levels. Compared with wild-type DAOY cells, AhR shRNA DAOY cells displayed an impaired G(1)-to-S cell cycle transition, decreased DNA synthesis, and reduced proliferation. Furthermore, these cell cycle perturbations were correlated with decreased levels of the pro-proliferative gene Hes1 and increased levels of the cell cycle inhibitor p27(kip1). Supplementation experiments with human AhR restored the proliferative activity in AhR shRNA DAOY cells. Taken together, our data show that the AhR promotes proliferation of MB cells, suggesting that this pathway should be considered as a potential therapeutic target for MB treatment.

Recent epidemiologic studies have demonstrated a link between organochlorine and pesticide exposure to an enhanced risk for neurodegenerative disorders such as Parkinson’s disease (PD). A common biological phenomenon underlying cell injury associated with both polychlorinated biphenyl (PCB) exposure and dopaminergic neurodegeneration during aging is oxidative stress (OS). In this study, we tested the hypothesis that oral PCB exposure, via food ingestion, impairs dopamine systems in the adult murine brain. We determined whether PCB exposure was associated with OS in dopaminergic neurons, a population of cells that selectively degenerate in PD. After 4 weeks of oral exposure to the PCB mixture Aroclor 1254, several congeners, mostly ortho substituted, accumulated throughout the brain. Significant increases in locomotor activity were observed within 2 weeks, which persisted after cessation of PCB exposure. Stereologic analyses revealed a significant loss of dopaminergic neurons within the substantia nigra and ventral tegmental area. However, striatal dopamine levels were elevated, suggesting that compensatory mechanisms exist to maintain dopamine homeostasis, which could contribute to the observed increases in locomotor activity following PCB exposure. Biochemical experiments revealed alterations in OS markers, including increases in SOD and HO-1 levels and the presence of oxidatively modified lipids and proteins. These findings were accompanied by elevated iron levels within the striatal and midbrain regions, perhaps due to the observed dysregulation of transferrin receptors and ferritin levels following PCB exposure. In this study, we suggest that both OS and the uncoupling of iron regulation contribute to dopamine neuron degeneration and hyperactivity following PCB exposure.

Thirty six undergraduate students participated in a study that compared inattentional blindness (IB) with situation awareness (SA), working memory (WM), and driving hazard detection. To test IB, participants watched the Invisible Gorilla video (Simon & Chabris, 1999) of a person dressed in a gorilla costume walking between players passing a basketball. Participants also watched a string of driving videos (automobile and motorcycle) from the driver's perspective. Participants answered SA questions (relevant to the driving task and irrelevant to the driving task) and identified driving hazards throughout the video. Participants' SA and hazard detection performance were compared to their IB (did see the gorilla, or did not see the gorilla), and their performance on counting completed basketball passes. In addition, participants' working memory (WM) and short-term memory (STM) were measured. Susceptibility to IB indicated longer response times for irrelevant questions related to the driving task when compared to relevant driving questions. Those who did not see the gorilla (DNS) also took longer to respond to irrelevant questions than those who did see the gorilla (DS). However, the two groups did not differ in the time taken to answer relevant driving SA questions. The DNS group outperformed the DS group on hazard detection performance. Finally, a trend suggested that individuals in the DNS group who had high WM were more accurate in pass counts. Conversely, individuals in the DNS group who had low WM were least accurate in pass counts. Keywords: Driver distraction; Language: en

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.

The widespread environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been linked to developmental neurotoxicity associated with abnormal cerebellar maturation in both humans and rodents. TCDD mediates toxicity via binding to the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of xenobiotic metabolizing enzymes and growth regulatory molecules. Our previous studies demonstrated that cerebellar granule neuron precursor cells (GNPs) express transcriptionally active AhR during critical developmental periods. TCDD exposure also impaired GNP proliferation and survival in vitro. Therefore, this study tested the hypothesis that TCDD exposure disrupts cerebellar development by interfering with GNP differentiation. In vivo experiments indicated that TCDD exposure on postnatal day (PND) 6 resulted in increased expression of a mitotic marker and increased thickness of the external granule layer (EGL) on PND10. Expression of the early differentiation marker TAG-1 was also more pronounced in postmitotic, premigratory granule neurons of the EGL, and increased apoptosis of GNPs was observed. On PND21, expression of the late GNP differentiation marker GABA(A alpha 6) receptor (GABAR(A alpha 6)) and total estimated cell numbers were both reduced following exposure on PND6. Studies in unexposed adult AhR(-/-) mice revealed lower GABAR(A alpha 6) levels and DNA content. In vitro studies showed elevated expression of the early differentiation marker p27/Kip1 and the GABAR(A alpha 6) in GNPs following TCDD exposure, and the expression patterns of proteins related to granule cell neurite outgrowth, beta III-tubulin and polysialic acid neural cell adhesion molecule, were consistent with enhanced neuroblast differentiation. Together, our data suggest that TCDD disrupts a normal physiological role of AhR, resulting in compromised GNP maturation and neuroblast survival, which impacts final cell number in the cerebellum.

The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic-helix-loop-helix/PER-ARNT-SIM(PAS) transcription factor superfamily that also mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence suggests that AhR influences the development of many tissues, including the central nervous system. Our previous studies suggest that sustained AhR activation by TCDD and/or AhR deletion disrupts cerebellar granule neuron precursor (GNP) development. In the current study, to determine whether endogenous AhR controls GNP development in a cell-autonomous manner, we created a GNP-specific AhR deletion mouse, AhR(fx/fx) /Math1(CRE/+) (AhR CKO). Selective AhR deletion in GNPs produced abnormalities in proliferation and differentiation. Specifically, fewer GNPs were engaged in S-phase, as demonstrated by ∼25% reductions in thymidine (in vitro) and Bromodeoxyuridine (in vivo) incorporation. Furthermore, total granule neuron numbers in the internal granule layer at PND21 and PND60 were diminished in AhR conditional knockout (CKO) mice compared with controls. Conversely, differentiation was enhanced, including ∼40% increase in neurite outgrowth and 50% increase in GABARα6 receptor expression in deletion mutants. Our results suggest that AhR activity plays a role in regulating granule neuron number and differentiation, possibly by coordinating this GNP developmental transition. These studies provide novel insights for understanding the normal roles of AhR signaling during cerebellar granule cell neurogenesis and may have important implications for the effects of environmental factors in cerebellar dysgenesis.

Site-specific gene editing combined with autologous stem cell transplantation holds tremendous potential to treat both inherited and acquired diseases. However, the translational applications of gene editing are limited by the challenge of achieving sufficient levels of gene targeting in human primary cells. Recent advances in gene editing, including use of the CRISPR-Cas9 system, nuclease-resistant chemically modified small guide RNAs (sgRNAs), and serotype-optimized AAV delivery of donor repair DNA templates, are enabling clinically relevant levels of gene editing in human primary cells. Here, we build upon these advances to develop a gene editing strategy to treat the immune-dysregulation polyendocrinopathy-enteropathy-X-linked (IPEX) syndrome, a severe primary immune deficiency manifesting with life-threatening multi-organ autoimmunity in children. IPEX is caused by mutations in the forkhead box protein 3 gene (FOXP3), resulting in dysfunction of T regulatory cells (Tregs) and T effector cell lymphoproliferation. Due to the presence of disease causative mutations throughout the entire FOXP3 gene and the complex nature of FOXP3 regulatory elements, we designed a strategy to functionally correct the gene in IPEX patient cells using on-target, homology directed repair (HDR)-mediated insertion of the FOXP3 coding sequence into the gene locus. By targeting the FOXP3 gene using a CRISPR system comprised of Cas9 mRNA and chemically modified sgRNAs, we were able to attain high targeting frequencies, reaching 70-80% in human primary CD4+ T cells. We use this CRISPR system in combination with a repair donor DNA template delivered with AAV to demonstrate FOXP3 gene correction in human CD34+ hematopoietic stem and progenitor cells (HSPCs). In addition to gene correction of FOXP3, we use a similar strategy in parallel to knockout FOXP3 in wild-type human CD34+ HSPCs. We use the knockout HSPCs to generate a humanized mouse model of FOXP3 dysfunction, which enables us to study the pathophysiology of IPEX and to ultimately test the efficacy of gene-corrected Treg cell-based therapies. These results will help demonstrate the feasibility of FOXP3 gene editing, which we propose for the translational application of autologous transplant of Tregs and HSPCs as a therapy for IPEX syndrome.

Precise engineered nuclease-mediated gene correction via homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the power to transform curative therapies for monogenic diseases of the immune system. Sickle cell disease (SCD) is one of the most common monogenic diseases, affecting millions of people worldwide. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with SCD; however, immunocompatibility issues, graft-versus-host disease, and graft rejection are major roadblocks for efficacious therapy. In theory, the ideal curative strategy for SCD and most monogenic immune diseases is ex vivo gene correction of patient-derived HSPCs followed by autologous HSCT of a highly purified targeted population to avoid possible complications of competition between unedited and gene-targeted HSPCs in vivo. By supplying a homologous GFP-expressing HBB donor via recombinant adeno-associated virus serotype 6 (rAAV6) in combination with a double strand break created by the CRISPR/Cas9 system, we achieved HR frequencies of 15-49% of HSPCs, and more importantly, identified a population of HBB-targeted HSPCs (HSPCHBB) through a log-fold MFI increase in chromosomal transgene expression. Fluorescence activated cell sorting (FACS) of this population revealed consistent GFP expression in >95% of cells over two weeks in vitro in multiple CD34+ donors isolated from either bone marrow, cord blood or peripheral blood. Single-cell FACS of HSPCHBB into methylcellulose led to both myeloid and lymphoid colony formation, and on-target PCR analysis revealed >95% of these HSPCHBB clones had either a mono or biallelic-targeting event. Notably, a fraction of HSPCHBB displayed the CD34+/CD38-/CD90+/CD45RA-immunophenotype, suggesting successful targeting of long-term repopulating hematopoietic stem cells (LT-HSCs). Furthermore, HSPCHBB displayed long-term engraftment in the bone marrow of immunodeficient NSG mice at 12 weeks post-transplant, where we identified that ~70% of human cells were GFP+ and also produced both myeloid (CD33+) and lymphoid (CD19+) cell types, implying that the HSPCHBB population contains true LT-HSCs that can repopulate a functional immune system. Altogether, these proof-of-concept studies showed that by combining a homologous rAAV6 donor, the CRISPR/Cas9 system, and FACS, it is feasible to generate and enrich a highly purified population of gene-corrected HSPCs that include LT-HSC potentials.