Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines.

Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species.

T cells specific for α-myosin drive immunotherapy-related myocarditis.

Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to the utility of immune checkpoint inhibitors (ICIs) in anticancer therapy. The pathogenesis of ICI-associated myocarditis (ICI-MC) is poorly understood. Pdcd1Ctla4 mice recapitulate clinicopathological features of ICI-MC, including myocardial T cell infiltration.

Surveying Mechanisms of Immune Checkpoint Blockade from a VISTA.

Defining the mechanisms of action of immune checkpoint blockade therapies is essential for effectively designing combination therapeutic approaches and developing the next generation of immunotherapies. In this issue, Schaafsma and colleagues report that V-domain immunoglobulin suppressor of T-cell activation antagonism acts through mechanisms distinct from anti-CTLA-4 and anti-programmed cell death protein 1 via remodeling of the myeloid-cell compartment and modulating T-cell quiescence. See related article by Schaafsma et al.

Checkpoint Blockade + Chemotherapy: the Right Combination for AML?

An emerging strategy to enhance the efficacy of immune checkpoint blockade in relapsed/refractory cancers is increasing immunogenic cell death via combination with cytotoxic therapies. Understanding the effects of cytotoxic and immunotherapeutic agents on immune cell populations will enable improved mechanism-based design of combination therapies to maximum efficacy and minimum toxicity..

A Genetic Mouse Model Recapitulates Immune Checkpoint Inhibitor-Associated Myocarditis and Supports a Mechanism-Based Therapeutic Intervention.

Immune checkpoint inhibitors (ICI) targeting CTLA4 or PD-1/PD-L1 have transformed cancer therapy but are associated with immune-related adverse events, including myocarditis. Here, we report a robust preclinical mouse model of ICI-associated myocarditis in which monoallelic loss of in the context of complete genetic absence of leads to premature death in approximately half of mice. Premature death results from myocardial infiltration by T cells and macrophages and severe ECG abnormalities, closely recapitulating the clinical and pathologic hallmarks of ICI-associated myocarditis observed in patients.

Combination anti-CTLA-4 plus anti-PD-1 checkpoint blockade utilizes cellular mechanisms partially distinct from monotherapies.

Immune checkpoint blockade therapy targets T cell-negative costimulatory molecules such as cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1). Combination anti-CTLA-4 and anti-PD-1 blockade therapy has enhanced efficacy, but it remains unclear through what mechanisms such effects are mediated. A critical question is whether combination therapy targets and modulates the same T cell populations as monotherapies.

Negative Co-stimulation Constrains T Cell Differentiation by Imposing Boundaries on Possible Cell States.

Co-stimulation regulates T cell activation, but it remains unclear whether co-stimulatory pathways also control T cell differentiation. We used mass cytometry to profile T cells generated in the genetic absence of the negative co-stimulatory molecules CTLA-4 and PD-1. Our data indicate that negative co-stimulation constrains the possible cell states that peripheral T cells can acquire.

Inhibition of spinal 15-LOX-1 attenuates TLR4-dependent, nonsteroidal anti-inflammatory drug-unresponsive hyperalgesia in male rats.

Although nonsteroidal anti-inflammatory drugs are the first line of therapeutics for the treatment of mild to moderate somatic pain, they are not generally considered to be effective for neuropathic pain. In the current study, direct activation of spinal Toll-like 4 receptors (TLR4) by the intrathecal (IT) administration of KDO2 lipid A (KLA), the active component of lipopolysaccharide, elicits a robust tactile allodynia that is unresponsive to cyclooxygenase inhibition, despite elevated expression of cyclooxygenase metabolites in the spinal cord. Intrathecal KLA increases 12-lipoxygenase-mediated hepoxilin production in the lumbar spinal cord, concurrent with expression of the tactile allodynia.

Fundamental Mechanisms of Immune Checkpoint Blockade Therapy.

Immune checkpoint blockade is able to induce durable responses across multiple types of cancer, which has enabled the oncology community to begin to envision potentially curative therapeutic approaches. However, the remarkable responses to immunotherapies are currently limited to a minority of patients and indications, highlighting the need for more effective and novel approaches. Indeed, an extraordinary amount of preclinical and clinical investigation is exploring the therapeutic potential of negative and positive costimulatory molecules.

Distinct Cellular Mechanisms Underlie Anti-CTLA-4 and Anti-PD-1 Checkpoint Blockade.

Immune-checkpoint blockade is able to achieve durable responses in a subset of patients; however, we lack a satisfying comprehension of the underlying mechanisms of anti-CTLA-4- and anti-PD-1-induced tumor rejection. To address these issues, we utilized mass cytometry to comprehensively profile the effects of checkpoint blockade on tumor immune infiltrates in human melanoma and murine tumor models. These analyses reveal a spectrum of tumor-infiltrating T cell populations that are highly similar between tumor models and indicate that checkpoint blockade targets only specific subsets of tumor-infiltrating T cell populations.

Forcing through Tumor Metastasis: The Interplay between Tissue Rigidity and Epithelial-Mesenchymal Transition.

The mechanical properties of the tumor microenvironment have been increasingly recognized as potent modulators of cell behavior and function. In particular, tissue rigidity is functionally important during tumor progression. In this review, we survey recent advances in our understanding of the role of tissue rigidity in tumor progression and metastasis, the mechanisms by which mechanical cues integrate with biochemical signals from the microenvironment, and the underlying mechanotransduction pathways involved in tumor progression.

Matrix stiffness drives epithelial-mesenchymal transition and tumour metastasis through a TWIST1-G3BP2 mechanotransduction pathway.

Matrix stiffness potently regulates cellular behaviour in various biological contexts. In breast tumours, the presence of dense clusters of collagen fibrils indicates increased matrix stiffness and correlates with poor survival. It is unclear how mechanical inputs are transduced into transcriptional outputs to drive tumour progression.

Systematic analysis of rat 12/15-lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia.

Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15-LOX enzymes in HEK-293T cells and measured by LC-MS/MS the formation of HXB3, 12-HETE, 8-HETE, and 15-HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA-861, CDC, baicalein, and PD146176) vs.