Browsing by Subject "Gene editing"

Now showing 1 - 6 of 6
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    ASGCT 2023—Gene therapy is becoming medicine
    (Elsevier, 2023) Herzog, Roland W.; Bricker-Anthony, Courtney; Pediatrics, School of Medicine
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    Call for Papers: Expanding the Scale and Scope of Therapeutic Gene Editing
    (Elsevier, 2020-08-05) Kiem, Hans-Peter; Gaudelli, Nicole N.; Urnov, Fyodor D.; Frederickson, Robert M.; Pediatrics, School of Medicine
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    CASowary: CRISPR-Cas13 guide RNA predictor for transcript depletion
    (BMC, 2022) Krohannon, Alexander; Srivastava, Mansi; Rauch, Simone; Srivastava, Rajneesh; Dickinson, Bryan C.; Janga, Sarath Chandra; BioHealth Informatics, School of Informatics and Computing
    Background: Recent discovery of the gene editing system - CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (Cas), has resulted in its widespread use for improved understanding of a variety of biological systems. Cas13, a lesser studied Cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The Cas13 system utilizes base complementarity between a crRNA/sgRNA (crispr RNA or single guide RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Transcripts also exhibit complex three-dimensional structures and interact with an array of RBPs (RNA Binding Proteins), both of which may impact the effectiveness of transcript depletion of target sequences. However, our understanding of the features and corresponding methods which can predict whether a specific sgRNA will effectively knockdown a transcript is very limited. Results: Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA. We used publicly available RNA knockdown data from Cas13 characterization experiments for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information. Our model utilizes a Decision Tree architecture with a set of 112 sequence and target availability features, to classify sgRNA efficacy into one of four classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 70%. Additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting Cas system - CIRTS, confirming the robustness and reproducibility of our model's sgRNA predictions. Utilizing transcriptome wide protein occupancy map generated using POP-seq in HeLa cells against publicly available protein-RNA interaction map in Hek293 cells, we show that CASowary can predict high quality guides for numerous transcripts in a cell line specific manner. Conclusions: Application of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of therapeutic interventions linked with aberrations in RNA regulatory processes.
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    Harnessing the Potential of Chimeric Antigen Receptor T-Cell Therapy for the Treatment of T-Cell Malignancies: A Dare or Double Dare?
    (MDPI, 2022-12-08) Assi, Rita; Salman, Huda; Medicine, School of Medicine
    Historical standard of care treatments of T-cell malignancies generally entailed the use of cytotoxic and depleting approaches. These strategies are, however, poorly validated and record dismal long-term outcomes. More recently, the introduction and approval of chimeric antigen receptor (CAR)-T cell therapy has revolutionized the therapy of B-cell malignancies. Translating this success to the T-cell compartment has so far proven hazardous, entangled by risks of fratricide, T-cell aplasia, and product contamination by malignant cells. Several strategies have been utilized to overcome these challenges. These include the targeting of a selective cognate antigen exclusive to T-cells or a subset of T-cells, disruption of target antigen expression on CAR-T constructs, use of safety switches, non-viral transduction, and the introduction of allogeneic compounds and gene editing technologies. We herein overview these historical challenges and revisit the opportunities provided as potential solutions. An in-depth understanding of the tumor microenvironment is required to optimally harness the potential of the immune system to treat T-cell malignancies.
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    Porcine UL-16 Binding Protein 1 Is Not a Functional Ligand for the Human Natural Killer Cell Activating Receptor NKG2D
    (MDPI, 2023-11-07) Lopez, Kevin J.; Spence, John Paul; Li, Wei; Zhang, Wenjun; Wei, Barry; Cross-Najafi, Arthur A.; Butler, James R.; Cooper, David K. C.; Ekser, Burcin; Li, Ping; Surgery, School of Medicine
    Natural killer (NK) cells play a vital role in xenotransplantation rejection. One approach to induce NK cell immune tolerance is to prevent the NK cell-mediated direct killing of porcine cells by targeting the interaction of the activating receptor NKG2D and its ligands. However, the identity of porcine ligands for the human NKG2D receptor has remained elusive. Previous studies on porcine UL-16 binding protein 1 (pULBP-1) as a ligand for human NKG2D have yielded contradictory results. The goal of the present study was to clarify the role of pULBP-1 in the immune response and its interaction with human NKG2D receptor. To accomplish this, the CRISPR/Cas9 gene editing tool was employed to disrupt the porcine ULBP-1 gene in a 5-gene knockout porcine endothelial cell line (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin, 5GKO). A colony with two allele mutations in pULBP-1 was established as a 6-gene knockout pig cell line (6GKO). We found that pULBP-1-deficient pig cells exhibited a reduced binding capacity to human NKG2D-Fc, a recombinant chimera protein. However, the removal of ULBP-1 from porcine endothelial cells did not significantly impact human NK cell degranulation or cytotoxicity upon stimulation with the pig cells. These findings conclusively demonstrate that pULBP-1 is not a crucial ligand for initiating xenogeneic human NK cell activation.
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    Precise genome-editing in human diseases: mechanisms, strategies and applications
    (Springer Nature, 2024-02-26) Zheng, Yanjiang; Li, Yifei; Zhou, Kaiyu; Li, Tiange; VanDusen, Nathan J.; Hua, Yimin; Pediatrics, School of Medicine
    Precise genome-editing platforms are versatile tools for generating specific, site-directed DNA insertions, deletions, and substitutions. The continuous enhancement of these tools has led to a revolution in the life sciences, which promises to deliver novel therapies for genetic disease. Precise genome-editing can be traced back to the 1950s with the discovery of DNA's double-helix and, after 70 years of development, has evolved from crude in vitro applications to a wide range of sophisticated capabilities, including in vivo applications. Nonetheless, precise genome-editing faces constraints such as modest efficiency, delivery challenges, and off-target effects. In this review, we explore precise genome-editing, with a focus on introduction of the landmark events in its history, various platforms, delivery systems, and applications. First, we discuss the landmark events in the history of precise genome-editing. Second, we describe the current state of precise genome-editing strategies and explain how these techniques offer unprecedented precision and versatility for modifying the human genome. Third, we introduce the current delivery systems used to deploy precise genome-editing components through DNA, RNA, and RNPs. Finally, we summarize the current applications of precise genome-editing in labeling endogenous genes, screening genetic variants, molecular recording, generating disease models, and gene therapy, including ex vivo therapy and in vivo therapy, and discuss potential future advances.