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Hearing loss (HL) affects approximately 20% of the global population and the treatments are currently limited to hearing aids and cochlea implants. Gene therapy offers a possibility to prevent or even cure HL. With the aim to optimize the adeno-associated virus (AAV) vector system for inner ear directed gene therapy, we generated AAV peptide display libraries based on the AAV1, AAV2 and AAV6 capsid backbones. All libraries present random unique 7-mer peptide inserts at variable region VIII of the capsid protein with diversities ranging from 80,000-622,000 (maximum likelihood estimate, MLE). We conducted high-throughput in vivo selection screens in the inner ear of adult mice, testing alternative administration routes that demand overcoming robust biological barriers. The target tissue is the organ of Corti with its crucial mechanosensory hair cells (HCs) of the inner ear, the supporting cells (SCs) and the underlying spiral ganglion neurones (SGNs). Distinct variants were found to be accumulated to up to 5% for AAV2-based variants and up to 2.5% for AAVl-derived capsids after two rounds of in vivo selection. Interestingly, some top variants were already accumulated for the AAV6 KO library after only one selection round. A total of 20 top candidates from the AAV1 and AAV2 libraries were produced as vectors. They outperform the parental serotypes and show diverse expression patterns in the adult mouse cochlea. Three promising variants had the ability to transduce outer HCs, a challenging cell type to infect, and many also targeted inner HCs. Almost half of the variants also strongly transduced all layers of the stria vascula-ris - a viable target tissue for the treatment of age-related HL - and the SGNs were targeted, with 4 variants being highly specific for SGNs. In addition, different intensities of fluorescent transgene expression suggest differential efficacy in delivery or vector uncoating within the cells of the inner ear. Thus, we report on a set of promising new AAV variants with distinct features developed by in vivo high throughput selection screens for improving inner ear directed gene therapy. [ABSTRACT FROM AUTHOR]

Introduction: Cochlear implantation induces trauma leading to immunological reactions compromising the performance of the implant. Vesicle-enriched secretome fractions (VSF) derived from human umbilical cord mesenchymal stromal cells is a new class of biological therapeutic targeting cochlear cells to modulate immunological processes. Previously, efficacy in spiral ganglion cell culture and attenuation of threshold shifts and protection of hair cells in vivo were shown. Safety, biodistribution and neuroprotective effects of VSF to maintain gross structural integrity of the cochlea after implantation trauma has been evaluated. Material and methods: Hearing guinea pigs (GP) were implanted with a cochlear implant followed by administration of VSF and hearing was assessed until sacrifice after 4 weeks. A long-term group of hearing GP received VSF with a six month follow up of monthly hearing measurement. A third group of hearing GP received labelled VSF investigating distribution of VSF in the inner ear 1-2 hours post application. All groups underwent detailed histological assessment. Results: Treatment with VSF improved hearing after cochlear implantation compared to control animals. Fibrosis 4 weeks post implantation was at similar level to control animals. Electrophysiological and histological findings of the long-term group revealed no adverse effects of VSF and ABR thresholds remained on a similar level compared to control. Positive fluorescent signal of labelled VSF was confirmed in cells of the inner ear. Conclusions: Uptake of labelled VSF into the guinea pig cochlea has been demonstrated and treatment with VSF seems to mediate immunological processes to a healthier state and maintain gross structural integrity. Application of VSF associated with cochlear implantation seems to be a safe and solid combination to prevent post implantation trauma and preserve residual hearing. This work was funded by Med-El, the Deutsche Forschungs-gemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy - EXC 2177/1 - Project ID 390895286 and by the European Regional Development Fund Interreg V-A Italia-Austria 2014-2020 (Project "EXOTHERA IT AT 1036") and the Project "ExtraNeu" from the State of Salzburg. [ABSTRACT FROM AUTHOR]

Hearing loss (HL) affects over 460 million people worldwide, significantly affecting quality of life. Genetic analysis has identified more than 150 causative monogenic genes for non-syndromic sensorineural hearing loss, presenting attractive targets for gene therapy interventions. These approaches also have the potential to enhance conventional treatment strategies. Particularly interesting in this regard are cochlear implants (CI), used for severe to profound HL. However, their efficacy is believed to depend on spiral ganglion neuron (SGN) survival. Recognizing the potential of neurotrophins to enhance SGN survival and improve CI outcomes, we here report on the development of a novel adeno-associated virus (AAV) vector optimized for transducing SGN, even in adult mice and at low vector doses. For this purpose, the capsid was engineered to display a heptamer peptide, which was previously derived from a phage library screen. Structure-focused modeling of our novel vector Var9 indicated a clear change in cell attachment receptor binding due to peptide insertion compared to its parental serotype AAV2. Predictions were confirmed using affinity chromatography and competition assays. Interestingly, Var9 demonstrated faster transgene expression in HEI-OC1 cells, a murine otic progenitor cell line, despite significantly lower entry efficiency, indicating enhanced intracellular processing of the vector. Subsequent IF-FISH analysis at single-cell level as well as our indirect uncoating assay revealed that Var9 vectors clearly outperform AAV2 vectors regarding kinetics and level of uncoating (3-fold), i.e. release of their genome from the capsid, a prerequisite for transgene transcription. Finally, in a neurotrophic gene therapy approach, Var9 effectively prevented SGN degeneration by overexpressing BDNF in SGN of deafened mice. Histological analysis of the cochlea revealed remarkable protective effects of SGN comparable to untreated control levels in all cochlear turns of mice treated with Var9-BDNF. In conclusion, our novel AAV vector demonstrated superior properties crucial for efficient SGN transduction and will be further refined for clinical applications, aiming to enhance neural survival and improve outcomes for cochlear gene therapy in CI recipients. [ABSTRACT FROM AUTHOR]