Paediatrics (RCH) - Research Publications

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Author: Trapani, Joseph × Author: Voskoboinik, Ilia × Type: Journal Article ×

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    Severely impaired CTL killing is a feature of the neurological disorder Niemann-Pick disease type C1
    Castiblanco, D ; Rudd-Schmidt, JA ; Noori, T ; Sutton, VR ; Hung, YH ; Flinsenberg, TWH ; Hodel, AW ; Young, ND ; Smith, N ; Bratkovic, D ; Peters, H ; Walterfang, M ; Trapani, JA ; Brennan, AJ ; Voskoboinik, I (AMER SOC HEMATOLOGY, 2022-03-24)
    Niemann-Pick disease type C1 (NP-C1) is a rare lysosomal storage disorder resulting from mutations in an endolysosomal cholesterol transporter, NPC1. Despite typically presenting with pronounced neurological manifestations, NP-C1 also resembles long-term congenital immunodeficiencies that arise from impairment of cytotoxic T lymphocyte (CTL) effector function. CTLs kill their targets through exocytosis of the contents of lysosome-like secretory cytotoxic granules (CGs) that store and ultimately release the essential pore-forming protein perforin and proapoptotic serine proteases, granzymes, into the synapse formed between the CTL and target cell. We discovered that NPC1 deficiency increases CG lipid burden, impairs autophagic flux through stalled trafficking of the transcription factor EB (TFEB), and dramatically reduces CTL cytotoxicity. Using a variety of immunological and cell biological techniques, we found that the cytotoxic defect arises specifically from impaired perforin pore formation. We demonstrated defects of CTL function of varying severity in patients with NP-C1, with the greatest losses of function associated with the most florid and/or earliest disease presentations. Remarkably, perforin function and CTL cytotoxicity were restored in vitro by promoting lipid clearance with therapeutic 2-hydroxypropyl-β-cyclodextrin; however, restoration of autophagy through TFEB overexpression was ineffective. Overall, our study revealed that NPC1 deficiency has a deleterious impact on CTL (but not natural killer cell) cytotoxicity that, in the long term, may predispose patients with NP-C1 to atypical infections and impaired immune surveillance more generally.
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    Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance
    Fareh, M ; Zhao, W ; Hu, W ; Casan, JML ; Kumar, A ; Symons, J ; Zerbato, JM ; Fong, D ; Voskoboinik, I ; Ekert, PG ; Rudraraju, R ; Purcell, DFJ ; Lewin, SR ; Trapani, JA (NATURE PORTFOLIO, 2021-07-13)
    The recent dramatic appearance of variants of concern of SARS-coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape from host immunity and antiviral therapeutics. Here, we employ genome-wide computational prediction and single-nucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus-free models. Further, optimized and multiplexed Cas13b CRISPR RNAs (crRNAs) suppress viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including the recently emerging dominant variant of concern B.1.1.7. The comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches does not impair the capacity of a potent single crRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 strains in infected mammalian cells, including the Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint for antiviral drug development to suppress and prevent a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.