In this dissertation, I study strategic communication, in which a sender strategically discloses information to persuade a receiver to take an action favorable to the sender. I study two models of ...constrained communication. The first one generalizes the standard Bayesian Persuasion model to allow for the receiver's strategic behavior. The second one, joint work with Nour Chalhoub, studies a dynamic information disclosure under the assumption that the sender can only lie in one direction, by under-reporting the state, but never over-reporting it. The constraints in each model are intuitive for real-life application and lead to results that are of substantial difference from the results of the standard settings.
A study of the democratic culture is presented from the variable participation in the electoral context of the province of Cañar, Ecuador, considering 2009 as the base year. The purpose is to know ...the behavior about voter participation in the electoral processes of the last decade, from the systematic and methodical description of a collection of historical data. Theoretical approaches to democratic culture and studies applied to the research phenomenon were systematized. While, in 2019, participation increased to 53.65%, in Biblián; and, 43.24%, in Déleg Keywords: Participation, democratic culture, electoral process, absenteeism, Ecuador. 1.Introducción La Constitución de la República del Ecuador, en el artículo i consagra: "El Ecuador es un Estado constitucional de derechos y justicia, social, democrático, soberano, independiente, unitario, intercultural, plurinacional y laico (...)
We describe CALITAS, a CRISPR-Cas-aware aligner and integrated off-target search algorithm. CALITAS uses a modified and CRISPR-tuned version of the Needleman-Wunsch algorithm. It supports an ...unlimited number of mismatches and gaps and allows protospacer adjacent motif (PAM) mismatches or PAMless searches. CALITAS also includes an exhaustive search routine to scan genomes and genome variants provided with a standard Variant Call Format file. By default, CALITAS returns a single best alignment for a given off-target site, which is a significant improvement compared to other off-target algorithms, and it enables off-targets to be referenced directly using alignment coordinates. We validate and compare CALITAS using a selected set of target sites, as well as experimentally derived specificity data sets. In summary, CALITAS is a new tool for precise and relevant alignments and identification of candidate off-target sites across a genome. We believe it is the state of the art for CRISPR-Cas specificity assessments.
Multiplexed genome editing with DNA endonucleases has broad application, including for cellular therapies, but chromosomal translocations, natural byproducts of inducing simultaneous genomic breaks, ...have not been explored in detail. Here we apply various CRISPR-Cas nucleases to edit the T cell receptor alpha and beta 2 microglobulin genes in human primary T cells and comprehensively evaluate the frequency and stability of the resulting translocations. A thorough translocation frequency analysis using three orthogonal methods (droplet digital PCR, unidirectional sequencing, and metaphase fluorescence
hybridization) yielded comparable results and an overall translocation rate of ∼7% between two simultaneous CRISPR-Cas9 induced edits. In addition, we show that chromosomal translocations can be reduced when using different nuclease combinations, or by the presence of a homologous single stranded oligo donor for multiplexed genome editing. Importantly, the two different approaches for translocation reduction are compatible with cell therapy applications.
Following DNA replication, eukaryotic cells must biorient all sister chromatids prior to cohesion cleavage at anaphase. In animal cells, sister chromatids gradually biorient during prometaphase, but ...current models of mitosis in S. cerevisiae assume that biorientation is established shortly after S phase. This assumption is based on the observation of a bilobed distribution of yeast kinetochores early in mitosis and suggests fundamental differences between yeast mitosis and mitosis in animal cells. By applying super-resolution imaging methods, we show that yeast and animal cells share the key property of gradual and stochastic chromosome biorientation. The characteristic bilobed distribution of yeast kinetochores, hitherto considered synonymous for biorientation, arises from kinetochores in mixed attachment states to microtubules, the length of which discriminates bioriented from syntelic attachments. Our results offer a revised view of mitotic progression in S. cerevisiae that augments the relevance of mechanistic information obtained in this powerful genetic system for mammalian mitosis.
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•S. cerevisiae chromosomes biorient in a stochastic process until anaphase onset•Microtubule length control discriminates bioriented from syntelic attachments•Bilobed kinetochore distribution is not synonymous with biorientation
Super-resolution imaging approaches debunk an assumption that yeasts biorient their chromosomes with different kinetics than plant and animal cells.
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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.
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.
The use of genetically engineered T cells expressing chimeric antigen receptors (CAR) against CD19 or BCMA to treat hematologic malignancies has demonstrated promising results in clinical trials. ...However, it is possible that the efficacy of CAR T cells could be limited in some clinical indications due to suppressive factors in the tumor microenvironment. Immunosuppressive cytokines (e.g., IL-6, IL-10 and TGFβ) provide a favorable environment for tumor growth and inhibit the tumoricidal activity of the endogenous T cells. In particular, transforming growth factor β (TGFβ) has been shown to suppress T-cell proliferation, inhibit the maturation of T helper cells and reduce their effector functions. Elevated levels of TGFβ have been detected in many human cancers, including multiple myeloma (MM). Here, we sought to modulate the activity of CAR T cell-based immunotherapy for MM by inhibiting TGFβ signaling in BCMA-specific CAR T cells (Budha et al., Cancer Cell, 2012).
We evaluated two strategies to modulate the TGFβ pathway: (1) knocking out TGFβ Receptor II (TGFβRII) using CRISPR gene editing and (2) overexpressing a dominant negative TGFβRII in BCMA targeted CAR T cells. TGFβRII modulation inhibited TGFβ-dependent Smad2/3 phosphorylation. TGFβRII-modulated BCMA CAR T cells were insensitive to TGFβ-mediated inhibition of proliferation, cell cytotoxicity and cytokine production, both IFNg and granzyme B, in the context of primary and/or repeat antigen stimulation with BCMA+ve MM cell lines in vitro . We further evaluated the gene expression signatures of BCMA CAR T cells isolated from tumor and spleen in a NSG mouse xenograft model subcutaneously inoculated with myeloma cell line (RPMI8226). TGFβ pathway gene expression changes indicating TGFβ exposure were observed in BCMA CAR T cells isolated from tumor, but not spleen, suggesting that BCMA CAR T cells are encountering a TGFβ-enriched TME. However, both TGFβ pathway modulating strategies prevented the development of the TGFβ-induced gene expression phenotype.
Together, these data suggest that inhibition of TGFβRII signaling may potentiate CAR T activity by directly inhibiting immunosuppressive effects of TGFβ in the tumor microenvironment. By specifically targeting TGFβ signaling in CAR T cells, it may be possible to avoid side effects associated with systemic administration of TGFβ antagonists. Gene editing to remove TGFβRII expression or overexpressing dominant negative TGFβ RII could modulate the activity of BCMA CAR T therapy in MM.
Vong:Juno Therapeutics: Employment, Equity Ownership. Nye:Juno Therapeutics: Employment, Equity Ownership. Hause:Juno Therapeutics: Employment, Equity Ownership. Clouser:Juno Therapeutics: Employment, Equity Ownership. Jones:Juno Therapeutics: Employment, Equity Ownership. Burleigh:Juno Therapeutics: Employment, Equity Ownership. Borges:Editas Medicine: Employment, Equity Ownership. Chin:Editas Medicine: Employment, Equity Ownership. Marco:Editas Medicine: Employment, Equity Ownership. Barrera:Editas Medicine: Employment, Equity Ownership. Da Silva:Editas Medicine: Employment, Equity Ownership. Harbinski:Editas Medicine: Employment, Equity Ownership. Giannoukos:Editas Medicine: Employment, Equity Ownership. Dhanapal:Editas Medicine: Employment, Equity Ownership. Jiang:Juno Therapeutics: Employment, Equity Ownership. Salmon:Juno Therapeutics: Employment, Equity Ownership. Wilson:Editas Medicine: Employment, Equity Ownership. Myer:Editas Medicine: Employment, Equity Ownership. Welstead:Editas Medicine: Employment, Equity Ownership. Bond:Juno Therapeutics: Employment, Equity Ownership. Sather:Juno Therapeutics: Employment, Equity Ownership.
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