Background The tumor-selective human adenovirus Delta24-RGD is currently under investigation in phase II clinical trials for patients with recurrent glioblastoma (GBM). To improve treatments for patients with GBM, we explored the potential of combining Delta24-RGD with antibodies targeting immune checkpoints. Methods C57BL/6 mice were intracranially injected with GL261 cells and treated with a low dose of Delta24-RGD virus. The expression dynamics of 10 co-signaling molecules known to affect immune activity was assessed in tumor-infiltrating immune cells by flow cytometry after viral injection. The antitumor activity was measured by tumor cell killing and IFNγ production in co-cultures. Efficacy of the combination viro-immunotherapy was tested in vitro and in the GL261 and CT2A orthotopic mouse GBM models. Patient-derived GBM cell cultures were treated with Delta24-RGD to assess changes in PD-L1 expression induced by virus infection. Results Delta24-RGD therapy increased intratumoral CD8+ T cells expressing Inducible T-cell co-stimulator (ICOS) and PD-1. Functionality assays confirmed a significant positive correlation between tumor cell lysis and IFNγ production in ex vivo cultures (Spearman r = 0.9524; P < .01). Co-cultures significantly increased IFNγ production upon treatment with PD-1 blocking antibodies. In vivo, combination therapy with low-dose Delta24-RGD and anti-PD-1 antibodies significantly improved outcome compared to single-agent therapy in both syngeneic mouse glioma models and increased PD-1+ tumor-infiltrating CD8+ T cells. Delta24-RGD infection induced tumor-specific changes in PD-L1 expression in primary GBM cell cultures. Conclusions This study demonstrates the potential of using low-dose Delta24-RGD therapy to sensitize glioma for combination with anti-PD-1 antibody therapy.

Melanocytes are present in various parts of the inner ear, including the stria vascularis in the cochlea and the dark cell areas in the vestibular organs, where they contribute to endolymph homeostasis. Developmental studies describing the distribution of vestibular melanocytes are scarce, especially in humans. In this study, we investigated the distribution and maturation of the vestibular melanocytes in relation to the developing dark cell epithelium in inner ear specimens from week 5 to week 14 of development and in surgical specimens of the adult ampulla. Vestibular melanocytes were located around the utricle and the ampullae of the semicircular canals before week 7 and were first seen underneath the transitional zones and dark cell areas between week 8 and week 10. At week 10, melanocytes made intimate contact with epithelial cells, interrupting the local basement membrane with their dendritic processes. At week 11, most melanocytes were positioned under the dark cell epithelia. No melanocytes were seen around or in the saccule during all investigated developmental stages. The dark cell areas gradually matured and showed an adult immunohistochemical profile of the characteristic ion transporter protein Na+/K+‐ATPase α1 by week 14. Furthermore, we investigated the expression of the migration‐related proteins ECAD, PCAD, KIT, and KITLG in melanocytes and dark cell epithelium. This is the first study to describe the spatiotemporal distribution of vestibular melanocytes during the human development and thereby contributes to understanding normal vestibular function and pathophysiological mechanisms underlying vestibular disorders.

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Non-Syndromic Hereditary Hearing Loss (NSHHL) is a genetically heterogeneous sensory disorder with about 120 genes already associated. Through exome sequencing and data aggregation, we identified a family with six affected individuals and one unrelated NSHHL patient with predicted-to-be deleterious missense variants in USP48. We also uncovered an eighth patient presenting unilateral cochlear nerve aplasia and a de novo splice variant in the same gene. USP48 encodes a ubiquitin carboxyl-terminal hydrolase under evolutionary constraint. Pathogenicity of the variants is supported by in vitro assays that showed that the mutated proteins are unable to hydrolyze tetra-ubiquitin. Correspondingly, three-dimensional representation of the protein containing the familial missense variant affects a loop that controls binding to ubiquitin. Consistent with a contribution of USP48 to auditory function, immunohistology showed that the encoded protein is expressed in the developing human inner ear, specifically in the spiral ganglion neurons, outer sulcus, interdental cells of the spiral limbus, stria vascularis, Reissner’s membrane, and in the transient Kolliker’s organ that is essential for auditory development. Engineered zebrafish knocked-down for usp48, the USP48 ortholog, presented with a delayed development of primary motor neurons, less developed statoacoustic neurons innervating the ears, decreased swimming velocity and circling swimming behavior indicative of vestibular dysfunction and hearing impairment. Corroboratingly, acoustic startle response assays revealed a significant decrease of auditory response of zebrafish lacking usp48 at 600 Hz and 800 Hz wavelengths. In conclusion, we describe a novel autosomal dominant NSHHL gene through a multipronged approach combining exome sequencing, animal modeling, immunohistology and molecular assays.

Numerous studies have shown the recovery of auditory function in mouse models of genetic hearing loss following AAV gene therapy, yet translation to the clinic has not yet been demonstrated. One limitation has been the lack of human inner ear cell lines or tissues for validating viral gene therapies. Cultured human inner ear tissue could help confirm viral tropism and efficacy for driving exogenous gene expression in targeted cell types, establish promoter efficacy and perhaps selectivity for targeted cells, confirm the expression of therapeutic constructs and the subcellular localization of therapeutic proteins, and address the potential cellular toxicity of vectors or exogenous constructs. To begin to address these questions, we developed an explant culture method using native human inner ear tissue excised at either fetal or adult stages. Inner ear sensory epithelia were cultured for four days and exposed to vectors encoding enhanced green fluorescent protein (eGFP). We focused on the synthetic AAV9-PHP.B capsid, which has been demonstrated to be efficient for driving eGFP expression in the sensory hair cells of mouse and non-human primate inner ears. We report that AAV9-PHP.B also drives eGFP expression in fetal cochlear hair cells and in fetal and adult vestibular hair cells in explants of human inner ear sensory epithelia, which suggests that both the experimental paradigm and the viral capsid may be valuable for translation to clinical application.