gene editing
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Mechanisms of antigen-specific T cell activation in MOGAD
PROJECT SUMMARY / ABSTRACT The overarching goal of this application is to train Dr. Carson E. Moseley, MD, PhD, who is a clinical neurologist and a research immunologist, to become an independent investigator studying and treating neuroimmunologic disorders. Myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is a recently described, severe, neuroinflammatory syndrome of the central nervous system (CNS) with no approved therapies. Although MOG-specific antibodies helped define the disease, MOG antibodies alone are not clearly pathogenic and our understanding of MOGAD immunopathology is limited. CD4+ T cells are a dominant lymphocyte population in MOGAD lesions, yet the targets of T cell responses to MOG and how T and B cells interact to drive pathogenic immune response in MOGAD are unknown. This proposal uses a complementary approach of human and mouse immunology along with new technologies in T cell repertoire mapping and genome editing to dissect MOG-specific CD4+ T cell responses in MOGAD. Additionally, it will use new models to investigate how B cells promote pathogenic T cell differentiation and select pathogenic T cell receptors. The proposed training plan involves mentored training, seminars, formal learning, and advising to ensure completion of the proposed research and Dr. Moseley’s career development. He will train at UCSF, which is an outstanding institute for research and environment for physician-scientists. He will receive training in human immunology and CRISPR-based gene editing technologies. He will be mentored by Dr. Scott Zamvil, a leader in identifying antigen-specific T cell responses in neuroimmunologic disorders, and co-mentored by Dr. Alexander Marson, an expert in CRISPR gene editing to understand lymphocyte function. This application will provide Dr. Moseley with the long-term skills needed to become an independent investigator leading efforts to study and treat neuroimmunologic disorders.
Multiplex single-cell chemical genomics to identify small molecule modulators of tumor cell-intrinsic immunogenicity in glioblastoma
PROJECT SUMMARY/ABSTRACT Glioblastoma multiforme is the most common and aggressive primary brain cancer. Despite a multimodal treatment regimen of surgical resection, chemotherapy, radiotherapy, and tumor-treating fields, most patients succumb to the disease within two years of diagnosis. Cancer immunotherapy strategies have emerged as a powerful tool for treating aggressive solid tumors such as melanoma and non-small cell lung cancer. However, current strategies have led to low response rates in glioblastoma, resulting from its low immunogenicity. The proposed research program aims to identify small molecules capable of increasing the immunogenicity of glioblastoma cells, focusing on altering gene expression programs associated with recognition by the immune system and the ability of cytotoxic immune cells to target glioblastoma for destruction. We will use highly multiplex chemical transcriptomic profiling to determine the molecular consequence of exposing glioblastoma neurosphere models to 3,792 small molecules, targeting the majority of cellular activities and clinically relevant drug targets as well as a collection of previously identified immunomodulators. We will then determine how each exposure alters the expression of gene programs associated with tumor cell immunogenicity and response to therapy, including the expression of genes associated with the recognition by the immune system and those associated with immune checkpoints, as well as programs more broadly correlated with resistance to anti-cancer therapies. Chemical hits that meet specific criteria will be subjected to a medicinal chemistry review to further classify compounds by their suitability for treating malignancies in the brain. We will then screen chemical hits to determine their ability to modulate immune-mediated tumor cell killing using tumor- immune cell co-culture. Lastly, we will leverage gene editing and flow cytometry to validate hits based on on- target molecular effects and further refine the mechanism of action by inspecting the ability of drugs to modulate immunogenic programs at the protein level. Our chemical genomics screens aim to provide crucial information regarding the link between pathway activity and immunomodulation in GBM, a critical step to guide future efforts in GBM immunotherapy. More broadly, our study will establish single-cell chemical genomics as a scalable platform for phenotype-based screening for preclinical prioritization of chemical modulators of complex transcriptional phenotypes and provide a framework for hit prioritization, establishment of pipeline robustness and hit validation in the context of single- cell chemical genomics screens.
Developing a novel technology for studying T cell differentiation in vivo
Summary CRISPR-based genetic screens have revolutionized our understanding of gene functions and molecular mechanisms across various biological processes. In the field of T cell biology, CRISPR screens have played a pivotal role in identifying genes that impact critical aspects, such as T cell development, differentiation, and function. However, traditional screens have struggled to distinguish genes with diverse mechanisms of action, necessitating further investigations. To address this challenge, researchers have harnessed the power of CRISPR screens combined with single-cell sequencing (scCRISPR-seq), enabling the simultaneous assessment of genetic perturbations and high-dimensional phenotypes at the single-cell level. While scCRISPR- seq has predominantly been performed in vitro using immortalized cell lines, its physiological relevance is limited due to oversimplified biological context and disparities compared to primary cells. This limitation highlights the urgent need for large-scale in vivo scCRISPR-seq with primary T cells. However, various challenges have discouraged its widespread adoption. The use of viral vectors for sgRNA delivery compromises physiological relevance, as the in vitro activation conditions fail to faithfully represent the intricate T cell priming process in vivo. Moreover, viral vector components and continuous Cas9 expression can trigger immunogenicity and cytotoxicity, leading to cell depletion and hindering long-term studies. Additionally, current scCRISPR-seq methods face technical limitations, including low editing efficiency and inadequate perturbation identity recovery rates, which impede efficient large-scale in vivo applications. Fortunately, recent advances in ribonucleoprotein complex (RNP) transfection have addressed many of these challenges. This cutting-edge technology enables efficient gene editing in primary T cells without the need for in vitro activation or permanent Cas9 expression. Leveraging the high editing efficiency of RNP transfection, the investigator’s team aims to develop a novel strategy for in vivo T cell CRISPR screens. This innovative approach involves arrayed RNP transfection and co- transfer of T cells that recognize the relevant antigens. Instead of traditional genetic barcodes, the strategy utilizes congenic markers (CD45.1/45.2 and CD90.1/CD90.2) from donor TCR transgenic T cells as "external barcodes." These markers facilitate the recovery of gene perturbation identity at the single-cell level through the application of CITE-seq. Importantly, this RNP-based strategy seamlessly integrates with existing single-cell sequencing protocols, enabling the comprehensive assessment of transcripts, epitopes, and chromatin accessibility simultaneously. To demonstrate the efficacy of this strategy, the team plans to develop two benchmarking approaches: RNP-CET-seq to investigate the role of TCR regulators in T cell exhaustion and RNP-CATE-seq to map the gene regulatory atlas of exhausted CD8 T cells. In summary, the proposed RNP- based scCRISPR-seq strategy overcomes the limitations of current approaches, enabling large-scale, multi- module in vivo genetic screens within a physiologically relevant context across various disease models.
Chromatin-Based Mechanisms Linking Transcriptional Dysregulation to Genome Instability in Neurodevelopmental Disorders.
PROJECT SUMMARY/ABSTRACT Neurons depend on a finely tuned interplay between chromatin regulation and genome maintenance, yet they are acutely vulnerable to DNA damage generated during activity-dependent transcription of long, synaptic genes. Disruption of this balance is increasingly recognized as a driver of neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD), intellectual disability, and epilepsy. High-confidence genetic studies converge on regulators of histone H3 lysine 4 (H3K4) methylation, such as the writers ASHIL and Klv1T2C and the eraser KDNISB, as recurrently mutated loci in NTIDs. The overarching goal of this study is to investigate how dysregulated H3K4 methylation compromises genome integrity in human neurons, thereby contributing to the pathogenesis of NDDs. The central, hypothesis is that coordinated II3K4 methylation safeguards neuronal genomes by maintaining an open chromatin architecture that permits the efficient detection and repair of transcription-coupled DNA lesions. The rationale/Or this study is to define the epigenetic control of DNA repair, which will illuminate a shared pathogenic hub across multiple ~I)D-linked genes. During the mentoredK99 phase, I will define how ASHIL, KMT2C, and KDM5B regulate chromatin structure and DNA repair at baseline and during transcriptional stress. Aim-1: I will use isogenic iPSC-derived cortical neurons with patient-relevant mutations or CRrSPRi knockdowns of these regulators, applying an integrated multi-omic pipeline: CUT&Tag and Micro-C to map H3K4 methylation and 3D chromatin topology. Aim-2: I will use Paired-Damage-seq, and CUT&RUN to chart oxidative lesions, repair synthesis, and recruitment of key repair factors; and RNA-seq to relate damage hotspots to altered gene expression. Aims l and 2 will be performed under the guidance of Dr. Lizarraga and Dr. Morrow, experts in the field of neurodevelopmental biology. My advisory team brings unique and complementary skills, enhancing my knowledge in 3D chromatin structure, transcription-coupled repair, gene editing, and multi-omics analysis. I will utilize these skills in the R00 phase (Aim 3), expanding the framework to include additional H3K4 regulators (e.g., LSD1, KMT2A) and broader neural lineages, thereby developing a comprehensive model. This study is innovative in its integration of single-cell D.NA damage mapping with chromatin topology and transcriptional profiling, enabling a direct and mechanistic connection between disrupted H3K4 methylation and genome instability. By uncovering how H3.K4 methylation prevents transcription-coupled genome instability in the developing brain, this research will address a critical gap in our understanding of NDD mechanisms. This award will enable me to launch an independent research program dedicated to determining mechanisms of chromatin-based processes that maintain genome stability in the developing human brain.
Rett syndrome, MECP2 and therapeutic strategies
The development of the iPS cell technology has revolutionized our ability to study development and diseases in defined in vitro cell culture systems. The talk will focus on Rett Syndrome and discuss two topics: (i) the use of gene editing as an approach to therapy and (ii) the role of MECP2 in gene expression (i) The mutation of the X-linked MECP2 gene is causative for the disease. In a female patient, every cell has a wt copy that is, however, in 50% of the cells located on the inactive X chromosome. We have used epigenetic gene editing tools to activate the wt MECP2 allele on the inactive X chromosome. (ii) MECP2 is thought to act as repressor of gene expression. I will present data which show that MECP2 binds to Pol II and acts as an activator for thousands of genes. The target genes are significantly enriched for Autism related genes. Our data challenge the established model of MECP2’s role in gene expression and suggest novel therapeutic approaches.
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