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Introduction Graft-versus-host disease (GVHD) is a severe complication of allogeneic hematopoietic cell transplantation. In its chronic form, GVHD causes prolonged post-transplant morbidity and significant mortality. There is a paucity of approved therapies and the current standard of care results in incomplete responses that leave significant unmet need for novel agents. Mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) is a bi-functional protein that acts as a signal-propagation scaffold and protease for selective innate and adaptive immune cell receptor signaling. The established link between MALT1 and receptor-mediated activation of multiple immune cell types involved in cGVHD argues for it as a therapeutic target for cGVHD. Furthermore, we have identified MALT1 signaling to be important in linking receptor-initiated signaling to changes in cellular metabolism necessary for immune cell effector function and propose that immunometabolic profiling of immune cells from cGVHD patients may provide novel biomarkers of both disease pathophysiology and associated MALT1 activity. Methods, Results, Conclusion To assess MALT1 as a novel drug target in cGVHD, we first characterized the effectiveness of allosteric inhibition on protease, scaffolding, and metabolic activity in primary human T cells. MALT1 inhibition of T-cell receptor (TCR) activated CD4 + T cells reduced protease activity by >90% and scaffolding activity by ∼50%, downstream of TCR activation. Consistent with disruption of the TCR signaling cascade, MALT1 inhibition reduced T H1 and T H17 cytokines, and the T FH cytokine IL-21 in a concentration dependent manner. B-cell receptor (BCR) driven proliferation of human B cells and C-type-lectin receptor and immune complex driven cytokine production from human macrophages were also inhibited. We next tested MALT1 inhibition in a sclerodermatous rodent model of cGVHD (scGVHD). Allogeneic bone marrow and splenocytes from LP/J donor mice were transferred to irradiated C57BL/6 recipient mice. Starting on day 21 after transfer, animals were dosed orally once daily with a MALT1 inhibitor, or twice daily with the JAK1/2 inhibitor ruxolitinib as a comparator. All animals were monitored daily for weight change, survival and GVHD score. On a modified scGVHD scoring scale, animals treated with a MALT1 inhibitor had significantly more progression-free survival throughout the study than vehicle or ruxolitinib-treated animals. No differential effect was observed on body weight and overall survival. MALT1 inhibitor treatment, but not ruxolitinib, reduced frequencies of splenic T FH cells and germinal center B cells compared with vehicle-treated mice. Importantly, MALT1 inhibition had no effect on the frequency or numbers of splenic regulatory T cells. Additionally, a delayed-type hypersensitivity (DTH) response was abrogated with MALT1 inhibition, showing that T-cell-driven responses in the skin, an important target tissue in cGVHD, can be abrogated with MALT1 blockade. Given recent studies implicating MALT1 activity as a link between immunoreceptor signaling and activation-dependent metabolic activity in T cells, we performed non-targeted metabolomics on plasma and spleens from scGVHD animals. Clustering of splenic metabolite intensities across treatment groups revealed a subset of metabolites responsive to MALT1 inhibition. This motivated us to explore the metabolic activity of cells from GVHD patients. By developing a method to infer metabolic pathway activity from transcriptional profiles of whole blood, we were able to utilize publicly available RNA-seq data. Using a data set containing 8 healthy controls and longitudinal samples from 25 GVHD patients, this approach segregated GVHD patients into four subsets, with the two largest subsets showing oppositional activity in most major metabolic pathways. Collectively, these data reveal a breadth of MALT1 activity across diverse cell types implicated in the initiation and progression of cGVHD and support development of small-molecule inhibitors of MALT1. These data also define novel heterogeneity among GHVD patients based on immunometabolic activity and motivate development of metabolic biomarkers of MALT1 activity for clinical use. Figure 1 Figure 1. Disclosures Dispirito: Rheos Medicines: Current Employment. Badur: Rheos Medicines: Current Employment. Biswas: Rheos Medicines: Current Employment. Camacho: Rheos Medicines: Current Employment. Chalishazar: Rheos Medicines: Current Employment. DeChristopher: Rheos Medicines: Current Employment. Sellers: Rheos Medicines: Current Employment. Steadman: Rheos Medicines: Current Employment. Soh: Rheos Medicines: Current Employment. Monroe: Rheos Medicines: Consultancy. Long: Rheos Medicines: Current Employment.

Beta hemoglobinopathies resulting from dysfunctional or deficient adult beta-globin expression are some of the most prevalent inherited blood disorders in the world. Upregulation of developmentally-silenced fetal gamma-globin would replace adult beta-globin to ameliorate disease symptoms. One of the approaches to reactivate fetal globin expression in erythroid cells is through gene editing by zinc finger or CRISPR-Cas9 nucleases to disrupt the expression of a transcription factor BCL11A, which mediates fetal globin silencing. As BCL11A-deficiency leads to hematopoietic stem cells (HSCs) defects, the current editing approaches target the BCL11A erythroid-enhancer region located in intron 2 of the BCL11A gene to selectively reduce BCL11A expression in erythroid cells. Instead of targeting BCL11A, we sought to identify novel cis-regulatory elements at the beta-globin locus for targeted gene editing to achieve fetal globin reactivation. From a lenti-CRISPR mediated saturated mutagenesis screen covering the beta-globin locus using Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP)-2 cells, multiple fetal hemoglobin (HbF)-inducing genomic domains were identified. Most of the hits were concentrated at the gamma-globin (HBG1/2) promoters, clustered at known hereditary persistence of fetal hemoglobin (HPFH) mutation hotspots. In-depth genotype to phenotype analysis further defined the indels responsible for HbF induction in these subdomains. We interrogated multiple families of nucleases and guide RNA (gRNA) combinations with or without single-stranded oligodeoxynucleotides (ssODN) to guide editing outcome. gRNAs were selected based on their HbF induction potential (up to 40%) when introduced into mobilized peripheral blood (mPB) CD34+ hematopoietic stem and progenitor cells (HSPCs) as ribonucleoprotein (RNP) complexes. HSPCs transfected with RNPs targeting either the BCL11A erythroid-enhancer or the HBG1/2 proximal regions were then injected into NBSGW mice to study the editing in SCID-repopulating cells (SRC) and their multilineage reconstitution potential. All groups achieved high levels of human chimerism (>70% hCD45+/hCD45+mCD45) and comparable monocytes, granulocytes, B lymphocytes, and hCD34+ HSPCs reconstitution. However, BCL11A-edited cells showed selective reduction in erythroid lineage (CD235a+) output, up to 4-fold lower than untreated or HBG1/2 promoter-edited HSPCs. Sequencing analysis from lineage-specific sorted cells further revealed reduced editing levels at BCL11A erythroid-specific enhancer in the erythroid compartment compared to unfractionated bone marrow (BM) or other human lineages (70% in erythroid vs. 90% in all other lineages). Furthermore, the nonproductive fraction of indels that did not disrupt the BCL11A GATAA motif was significantly enriched in erythroid cells (22% in erythroid vs. 8% in other lineages). Ex vivo erythroid cultures suggests BCL11A erythroid-enhancer editing may lead to slightly increased apoptosis during erythroid differentiation. In contrast, HBG1/2 promoter-edited cells had similar editing levels and indel patterns across all lineages with no significant lineage skewing. When chimeric BM from HBG1/2 promoter-edited groups were cultured in erythroid conditions, ex vivo-derived erythroid cells had significantly elevated levels of HbF compared to controls. When CD235a+ cells were sorted without further culture from chimeric BM of mice engrafted with HBG1/2 promoter-edited cells, significantly increased levels of HbF were detected by UPLC (up to 30%) compared to controls (~6%). Thus, long-term HSCs have been edited productively at the HBG1/2 promoters. These cells were able to generate erythroid progenitors that harbor HbF-inducing indels, which in turn, gave rise to erythroid cells in vivo with a clinically-relevant levels of HbF in a xenotransplantation model. Together, our data suggest that BCL11A-edited cells have an erythroid differentiation defect or survival disadvantage in NBSGW mouse model that warrants further investigation. In contrast, editing of the HBG1/2 promoters in mPB CD34+ cells achieved sustained HbF expression in erythroid lineage while maintaining multilineage differentiation potential. Targeting of the HBG1/2 promoters in HSPCs may be an attractive strategy for the development of potential gene editing medicines for beta hemoglobinopathies. Disclosures Chang: Editas Medicine Inc.: Employment, Equity Ownership. Sanchez:Editas Medicine Inc.: Employment, Equity Ownership. Heath:Editas Medicine Inc.: Employment, Equity Ownership. deDreuzy:Editas Medicine Inc.: Employment, Equity Ownership. Haskett:Editas Medicine Inc.: Employment, Equity Ownership. Vogelaar:Editas Medicine Inc.: Employment. Gogi:Editas Medicine Inc.: Employment, Equity Ownership. Da Silva:Editas Medicine Inc.: Employment, Equity Ownership. Wang:Editas Medicine Inc.: Employment, Equity Ownership. Sadowski:Editas Medicine Inc.: Employment, Equity Ownership. Gotta:Editas Medicine Inc.: Employment, Equity Ownership. Siwak:Editas Medicine Inc.: Employment, Equity Ownership. Viswanathan:Editas Medicine Inc.: Employment, Equity Ownership. Loveluck:Editas Medicine Inc.: Employment, Equity Ownership. Chao:Editas Medicine Inc.: Employment, Equity Ownership. Tillotson:Editas Medicine Inc.: Employment, Equity Ownership. Chalishazar:Editas Medicine Inc.: Employment, Equity Ownership. Dass:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Brennan:Editas Medicine Inc.: Employment, Equity Ownership. Tabbaa:Editas Medicine Inc.: Employment, Equity Ownership. Marco:Editas Medicine Inc.: Employment, Equity Ownership. Zuris:Editas Medicine Inc.: Employment, Equity Ownership. Reyon:Editas Medicine Inc.: Employment, Equity Ownership. Isik:Editas Medicine Inc.: Employment, Equity Ownership. Friedland:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Harbinski:Editas Medicine Inc.: Employment, Equity Ownership. Giannoukos:Editas Medicine Inc.: Employment, Equity Ownership. Teixeira:Editas Medicine Inc.: Employment, Equity Ownership. Wilson:Editas Medicine Inc.: Employment, Equity Ownership. Albright:Editas Medicine Inc.: Employment, Equity Ownership. Jiang:Editas Medicine Inc.: Employment, Equity Ownership.

Transplantation of genetically corrected autologous hematopoietic stem/progenitor cells (HSPCs) is an effective treatment for patients with inherited hematologic disease. Genome editing with CRISPR/Cas9 ribonucleoprotein (RNP) precisely modifies gene targets in human cells, including HSPCs. In order to translate this technology to a clinical setting, gene editing must be reproducible and efficient across multiple patient donors without compromising HSPC viability, multipotency, and long-term engraftment capability. To determine the reproducibility of Cas9 RNP mediated gene editing in HSPCs, human CD34+ cells obtained from 15 different patient donors (cord blood n=12, mobilized peripheral blood [mPB] n=3) were electroporated with S. pyogenes Cas9 RNP targeting the β-hemoglobin (HBB) or AAVS1 genetic locus. DNA sequence analysis of 15 separate experiments demonstrated that Cas9 supported up to 72% gene editing in cord blood CD34+ cells and up to 61% gene editing in adult mPB CD34+ cells (mean % editing by DNA sequencing: 57% ± 8%). Cas9 induced multiple modifications that comprise insertions and deletions at the HBB locus, and some of the lesions were repaired through an HDR repair mechanism that used the homologous sequences from the endogenous HBD gene as a template (Gene Conversion). Gene edited CD34+ cells retained viability and hematopoietic colony forming cell (CFC) activity ex vivo, with no significant differences between treatment groups or across donors. Indels were detected in one (BFU-E/GEMM: 50%-75%, CFU-GM/M: 50-66%) or both (BFU-E/GEMM: 12-38%, CFU-GM/M: 12-50%) alleles, at high frequencies in clonal erythroid and myeloid colonies (range of 63-100% of individual colonies assayed across experiments). To evaluate engraftment potential, both Cas9 modified (unsorted) and untreated control human CD34+ cells were transplanted into immunodeficient mice. Twelve weeks after transplantation, up to 34% human CD45+ cell engraftment was detected in the peripheral blood of recipients of Cas9 RNP treated CD34+ cells. There was no significant difference in the engraftment of Cas9 RNP treated and untreated control CD34+ cells which were detected in blood, spleen, and bone marrow. Human lymphoid, myeloid, and erythroid cells were detected in blood and hematopoietic organs with no difference in lineage distribution between cohorts. Analysis of the bone marrow from both groups revealed an average of 20% human CD45+ and 13% CD34+CD45+ cell engraftment long-term. Analysis of human gDNA revealed that up to 20% gene editing in the marrow of treated mice and edited cells were also detected in the blood and spleen. In summary, these data show that Cas9 RNP gene edited primary human CD34+ HSPCs retained the ability to engraft long-term and reconstitute hematopoiesis in vivo at levels higher than reported previously. These findings also show that electroporation of HSPCs with Cas9 RNP mediated highly reproducible gene editing levels across multiple donors and did not alter hematopoietic reconstitution or differentiation properties in comparison to untreated control CD34+ cells.