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New drugs that precisely target the immune mechanisms critical for cytotoxic T lymphocyte (CTL) and natural killer (NK) cell driven pathologies are desperately needed. In this perspective, we explore the cytolytic protein perforin as a target for therapeutic intervention. Perforin plays an indispensable role in CTL/NK killing and controls a range of immune pathologies, while being encoded by a single copy gene with no redundancy of function. An immunosuppressant targeting this protein would provide the first-ever therapy focused specifically on one of the principal cell death pathways contributing to allotransplant rejection and underpinning multiple autoimmune and postinfectious diseases. No drugs that selectively block perforin-dependent cell death are currently in clinical use, so this perspective will review published novel small molecule inhibitors, concluding with

Cytotoxic T lymphocytes (CTLs) and natural killer cells kill virus-infected and tumor cells through the polarized release of perforin and granzymes. Perforin is a pore-forming toxin that creates a lesion in the plasma membrane of the target cell through which granzymes enter the cytosol and initiate apoptosis. Endosomal sorting complexes required for transport (ESCRT) proteins are involved in the repair of small membrane wounds. We found that ESCRT proteins were precisely recruited in target cells to sites of CTL engagement immediately after perforin release. Inhibition of ESCRT machinery in cancer-derived cells enhanced their susceptibility to CTL-mediated killing. Thus, repair of perforin pores by ESCRT machinery limits granzyme entry into the cytosol, potentially enabling target cells to resist cytolytic attack.

Precision oncology programs can rapidly identify oncogenic gene fusions in individual patients1–3. However, despite their established oncogenic status, the vast majority of gene fusions remain ‘undruggable’ due to the lack of specific inhibitory molecules4, 5. Here, we establish PspCas13b, a poorly characterized programmable RNA nuclease, as a versatile tool to silence various oncogenic fusion transcripts. Our Single-Base Tiled crRNA screens (SiBTil), unbiased computational analysis, and comprehensive spacer-target mutagenesis revealed key determinants of PspCas13b activity. De novo design of crRNAs harbouring basepaired or mismatched guanosine bases at key spacer positions greatly enhances the silencing efficacy of otherwise inefficient crRNAs, expanding the targeting spectrum of this enzyme. We also reveal the interface between mismatch tolerance and intolerance, which unlocks an unexpected single-base precision targeting capability of this RNA nuclease. Notably, our de novo design principles enable potent and selective silencing of various gene fusion transcripts and their downstream oncogenic networks, without off-targeting of non-translocated variants that share extensive sequence homology. We demonstrate that PspCas13b targeting the breakpoint of fusion transcripts enables efficient suppression of ancestral and single-nucleotide mutants (e.g. BCR-ABL1 T315I) that often drive clinical cancer relapse. Collectively, this study provides new design principles for PspCas13b programming to specifically recognise and degrade any ‘undruggable’ fusion oncogenic transcript, thus providing a new conceptual framework for personalized oncology.

Second generation chimeric antigen receptor (CAR) T cells were used to treat patients with acute myeloid leukemia (AML) in a phase I clinical study. Autologous T cells were genetically modified to express a CAR which re-directed T cell effector function to the LeY tumor associated carbohydrate antigen on AML cells. CAR-T cell therapy safety, AML disease response, and CAR-T cell trafficking and persistence post-infusion were investigated. Five patients received GMP grade CAR-T cells (LeY-T). Post infusion, no patients experienced grade 3 or 4 toxicities. Patient AML responses to LeY-T cell infusion included a transient cytogenetic response and a reduction in peripheral blood leukemic blast count. In all patients, LeY-T cells trafficked thru peripheral blood, and persisted in the bone marrow. In one patient, leukemia cutis was associated with trafficking of the LeY-T cells to the skin at sites of AML blast infiltration. Despite LeY-T cells being present at the disease site, relapse with LeY-expressing AML blasts occurred in all patients (range 29 days to 23 months) post-infusion. Further studies indicated LeY-T cell CAR expression was downregulated post-infusion in vivo, this was also observed post-LeY antigen exposure and long term culture in vitro. This study provides important safety and feasibility data to support the application of CAR-T cell therapy to treat AML. Furthermore, we provide a potential mechanism for tumor escape from LeY-T cell surveillance in vivo.