AAV-mediated Gene Cocktails Enhance Supporting Cell Reprogramming and Hair Cell Regeneration.

Mammalian cochlear hair cells (HCs) are essential for hearing, and damage to HCs results in severe hearing impairment. Damaged HCs can be regenerated by neighboring supporting cells (SCs), thus the functional regeneration of HCs is the main goal for the restoration of auditory function in vivo. Here, cochlear SC trans-differentiation into outer and inner HC by the induced expression of the key transcription factors Atoh1 and its co-regulators Gfi1, Pou4f3, and Six1 (GPAS), which are necessary for SCs that are destined for HC development and maturation via the AAV-ie targeting the inner ear stem cells are successfully achieved. Single-cell nuclear sequencing and lineaging tracing results showed that the majority of new Atoh1-derived HCs are in a state of initiating differentiation, while GP (Gfi1, Pou4f3) and GPS (Gfi1, Pou4f3, and Six1) enhanced the Atoh1-induced new HCs into inner and outer HCs. Moreover, the patch-clamp analysis indicated that newborn inner HCs induced by GPAS forced expression have similar electrophysiological characteristics to those of native inner HCs. Also, GPAS can induce HC regeneration in the HC-damaged mice model. In summary, the study demonstrates that AAV-mediated co-regulation of multiple genes, such as GPAS, is an effective means to achieve functional HC regeneration in the mouse cochlea.

Pcolce2 overexpression promotes supporting cell reprogramming in the neonatal mouse cochlea.

Hair cell (HC) damage is a leading cause of sensorineural hearing loss, and in mammals supporting cells (SCs) are unable to divide and regenerate HCs after birth spontaneously. Procollagen C-endopeptidase enhancer 2 (Pcolce2), which encodes a glycoprotein that acts as a functional procollagen C protease enhancer, was screened as a candidate regulator of SC plasticity in our previous study. In the current study, we used adeno-associated virus (AAV)-ie (a newly developed adeno-associated virus that targets SCs) to overexpress Pcolce2 in SCs. AAV-Pcolce2 facilitated SC re-entry into the cell cycle both in cultured cochlear organoids and in the postnatal cochlea. In the neomycin-damaged model, regenerated HCs were detected after overexpression of Pcolce2, and these were derived from SCs that had re-entered the cell cycle. These findings reveal that Pcolce2 may serve as a therapeutic target for the regeneration of HCs to treat hearing loss.

AAV-mediated Gpm6b expression supports hair cell reprogramming.

Irreversible damage to hair cells (HCs) in the cochlea leads to hearing loss. Cochlear supporting cells (SCs) in the murine cochlea have the potential to differentiate into HCs. Neuron membrane glycoprotein M6B (Gpm6b) as a four-transmembrane protein is a potential regulator of HC regeneration according to our previous research. In this study, we found that AAV-ie-mediated Gpm6b overexpression promoted SC-derived organoid expansion. Enhanced Gpm6b prevented the normal decrease in SC plasticity as the cochlea develops by supporting cells re-entry cell cycle and facilitating the SC-to-HC transformation. Also, overexpression of Gpm6b in the organ of Corti through the round window membrane injection facilitated the trans-differentiation of Lgr5+ SCs into HCs. In conclusion, our results suggest that Gpm6b overexpression promotes HC regeneration and highlights a promising target for hearing repair using the inner ear stem cells combined with AAV.

Advancements and future prospects of adeno-associated virus-mediated gene therapy for sensorineural hearing loss.

Sensorineural hearing loss (SNHL), a highly prevalent sensory impairment, results from a multifaceted interaction of genetic and environmental factors. As we continually gain insights into the molecular basis of auditory development and the growing compendium of deafness genes identified, research on gene therapy for SNHL has significantly deepened. Adeno-associated virus (AAV), considered a relatively secure vector for gene therapy in clinical trials, can deliver various transgenes based on gene therapy strategies such as gene replacement, gene silencing, gene editing, or gene addition to alleviate diverse types of SNHL. This review delved into the preclinical advances in AAV-based gene therapy for SNHL, spanning hereditary and acquired types. Particular focus is placed on the dual-AAV construction method and its application, the vector delivery route of mouse inner ear models (local, systemic, fetal, and cerebrospinal fluid administration), and the significant considerations in transforming from AAV-based animal model inner ear gene therapy to clinical implementation.

Human papillomavirus prevalence and genotype distribution in Liaocheng men between 2016 and 2022.

Human papillomavirus (HPV) infection can lead to HPV-related cancer in men, including the anus, penile, and oropharyngeal cancers and precancerous lesions. This study retrospectively investigated HPV prevalence and genotype distribution in Liaocheng men between 2016 and 2022. The total HPV positive rate was 64.87% (2388/3681, 95% confidence interval [CI]: 63.32%-66.40%), where high risk (HR)-HPV and low risk (LR)-HPV accounted for 42.49% (1564/3681, 95% CI: 40.90%-44.09%) and 69.71% (2566/3681, 95% CI: 68.20%-71.17%), respectively. The mixed HPV infection rate of two and more genotypes was 35.72%. The infection rate of HR-HPV increased with the number of positive cases annually from 2016 (16.91%) to 2022 (46.59%). The most common HR-HPV genotypes were HPV16 (11.60%), HPV52 (6.95%), and HPV59 (6.28%), whereas the least common HR-HPV was HPV26. The most common LR-HPV genotypes were HPV6 (56.99%), HPV11 (23.79%), and HPV43 (6.37%). The 9 v HPV vaccine preventable for LR-HPV and HR-HPV accounted for 80.78% and 30.40%, respectively, in this study. Most HPV-positive patients aged 1-86 were in the 30-39 age group. This study confirmed that HPV prevalence in Liaocheng men was common and diverse. HPV16, HPV52, and HPV59 are widely distributed in Liaocheng men, and the male HR-HPV infection rate remained high in this region. Regarding public health and cancer prevention, it is recommended and effective to include the HPV vaccination in the national vaccination program for men.

AAV-Mediated Gene Therapy Restores Hearing in Patients with DFNB9 Deafness.

Mutations in OTOFERLIN (OTOF) lead to the autosomal recessive deafness 9 (DFNB9). The efficacy of adeno-associated virus (AAV)-mediated OTOF gene replacement therapy is extensively validated in Otof-deficient mice. However, the clinical safety and efficacy of AAV-OTOF is not reported. Here, AAV-OTOF is generated using good manufacturing practice and validated its efficacy and safety in mouse and non-human primates in order to determine the optimal injection dose, volume, and administration route for clinical trials. Subsequently, AAV-OTOF is delivered into one cochlea of a 5-year-old deaf patient and into the bilateral cochleae of an 8-year-old deaf patient with OTOF mutations. Obvious hearing improvement is detected by the auditory brainstem response (ABR) and the pure-tone audiometry (PTA) in these two patients. Hearing in the injected ear of the 5-year-old patient can be restored to the normal range at 1 month after AAV-OTOF injection, while the 8-year-old patient can hear the conversational sounds. Most importantly, the 5-year-old patient can hear and recognize speech only through the AAV-OTOF-injected ear. This study is the first to demonstrate the safety and efficacy of AAV-OTOF in patients, expands and optimizes current OTOF-related gene therapy and provides valuable information for further application of gene therapies for deafness.

Critical role of TPRN rings in the stereocilia for hearing.

Inner ear hair cells detect sound vibration through the deflection of mechanosensory stereocilia. Cytoplasmic protein TPRN has been shown to localize at the taper region of the stereocilia, and mutations in TPRN cause hereditary hearing loss through an unknown mechanism. Here, using biochemistry and dual stimulated emission depletion microscopy imaging, we show that the TPRN, together with its binding proteins CLIC5 and PTPRQ, forms concentric rings in the taper region of stereocilia. The disruption of TPRN rings, triggered by the competitive inhibition of the interaction of TPRN and CLIC5 or exogenous TPRN overexpression, leads to stereocilia degeneration and severe hearing loss. Most importantly, restoration of the TPRN rings can rescue the damaged auditory function of Tprn knockout mice by exogenously expressing TPRN at an appropriate level in HCs via promoter recombinant adeno-associated virus (AAV). In summary, our results reveal highly structured TPRN rings near the taper region of stereocilia that are crucial for stereocilia function and hearing. Also, TPRN ring restoration in stereocilia by AAV-Tprn effectively repairs damaged hearing, which lays the foundation for the clinical application of AAV-mediated gene therapy in patients with TPRN mutation.

Stem Cell-Based Hair Cell Regeneration and Therapy in the Inner Ear.

Hearing loss has become increasingly prevalent and causes considerable disability, thus gravely burdening the global economy. Irreversible loss of hair cells is a main cause of sensorineural hearing loss, and currently, the only relatively effective clinical treatments are limited to digital hearing equipment like cochlear implants and hearing aids, but these are of limited benefit in patients. It is therefore urgent to understand the mechanisms of damage repair in order to develop new neuroprotective strategies. At present, how to promote the regeneration of functional hair cells is a key scientific question in the field of hearing research. Multiple signaling pathways and transcriptional factors trigger the activation of hair cell progenitors and ensure the maturation of newborn hair cells, and in this article, we first review the principal mechanisms underlying hair cell reproduction. We then further discuss therapeutic strategies involving the co-regulation of multiple signaling pathways in order to induce effective functional hair cell regeneration after degeneration, and we summarize current achievements in hair cell regeneration. Lastly, we discuss potential future approaches, such as small molecule drugs and gene therapy, which might be applied for regenerating functional hair cells in the clinic.

Preclinical Efficacy And Safety Evaluation of AAV-OTOF in DFNB9 Mouse Model And Nonhuman Primate.

OTOF mutations are the principal causes of auditory neuropathy. There are reports on Otof-related gene therapy in mice, but there is no preclinical research on the drug evaluations. Here, Anc80L65 and the mouse hair cell-specific Myo15 promoter (mMyo15) are used to selectively and effectively deliver human OTOF to hair cells in mice and nonhuman primates to evaluate the efficacy and safety of OTOF gene therapy drugs. A new dual-AAV-OTOF-hybrid strategy to transfer full-length OTOF is generated, which can stably restore hearing in adult OTOFp.Q939*/Q939* mice with profound deafness, with the longest duration being at least 150 days, and the best therapeutic effect without difference in hearing from wild-type mice. An AAV microinjection method into the cochlea of cynomolgus monkeys without hearing impairment is further established and found the OTOF can be safely and effectively driven by the mMyo15 promoter in hair cells. In addition, the therapeutic dose of AAV drugs has no impact on normal hearing and does not cause significant systemic toxicity both in mouse and nonhuman primates. In summary, this study develops a potential gene therapy strategy for DFNB9 patients in the clinic and provides complete, standardized, and systematic research data for clinical research and application.

The role of Espin in the stereocilia regeneration and protection in Atoh1-overexpressed cochlear epithelium.

Hair cells (HCs) in mammals cannot spontaneously regenerate after damage. Atoh1 overexpression can promote HC regeneration in the postnatal cochlea, but the regenerated HCs do not possess the structural and functional characteristics of HCs in situ. The stereocilia on the apical surface of HCs are the first-level structure for sound conduction, and regeneration of functional stereocilia is the key basis for the reproduction of functional HCs. Espin, as an actin bundling protein, plays an important role in the development and structural maintenance of the stereocilia. Here, we found that the upregulation of Espin by AAV-ie was able to induced the aggregation of actin fibres in Atoh1-induced HCs in both cochlear organoids and explants. In addition, we found that persistent Atoh1 overexpression resulted in impaired stereocilia in both endogenous and newly formed HCs. In contrast, the forced expression of Espin in endogenous and regenerative HCs was able to eliminate the stereocilia damage caused by persistent Atoh1 overexpression. Our study shows that the enhanced expression of Espin can optimize the developmental process of stereocilia in Atoh1-induced HCs and can attenuate the damage to native HCs induced by Atoh1 overexpression. These results suggest an effective method to induce the maturation of stereocilia in regenerative HCs and pave the way for functional HC regeneration via supporting cell transdifferentiation.

AAV-Net1 facilitates the trans-differentiation of supporting cells into hair cells in the murine cochlea.

Mechanosensitive hair cells (HCs) in the cochlear sensory epithelium are critical for sound detection and transduction. Mammalian HCs in the cochlea undergo cytogenesis during embryonic development, and irreversible damage to hair cells postnatally is a major cause of deafness. During the development of the organ of Corti, HCs and supporting cells (SCs) originate from the same precursors. In the neonatal cochlea, damage to HCs activates adjacent SCs to act as HC precursors and to differentiate into new HCs. However, the plasticity of SCs to produce new HCs is gradually lost with cochlear development. Here, we delineate an essential role for the guanine nucleotide exchange factor Net1 in SC trans-differentiation into HCs. Net1 overexpression mediated by AAV-ie in SCs promoted cochlear organoid formation and HC differentiation under two and three-dimensional culture conditions. Also, AAV-Net1 enhanced SC proliferation in Lgr5-EGFPCreERT2 mice and HC generation as indicated by lineage tracing of HCs in the cochleae of Lgr5-EGFPCreERT2/Rosa26-tdTomatoloxp/loxp mice. We further found that the up-regulation of Wnt/ß-catenin and Notch signaling in AAV-Net1-transduced cochleae might be responsible for the SC proliferation and HC differentiation. Also, Net1 overexpression in SCs enhanced SC proliferation and HC regeneration and survival after HC damage by neomycin. Taken together, our study suggests that Net1 might serve as a potential target for HC regeneration and that AAV-mediated gene regulation may be a promising approach in stem cell-based therapy in hearing restoration.

Rps14 upregulation promotes inner ear progenitor proliferation and hair cell regeneration in the neonatal mouse cochlea.

Sensorineural hearing loss a result from hair cell damage, which is irreversible in mammals owing to the lack of hair cell regeneration, but recent researches have shown that Lgr5+ supporting cells are progenitors capable of regenerating hair cells. RPS14 (ribosomal protein S14) is a 40S ribosomal subunit component and is associated with erythrocyte differentiation, and in this study, we used a novel adeno-associated virus-inner ear system to upregulate Rps14 expression in cultured hair cell progenitors and observed an enhancement on their ability to proliferate and differentiate into hair cells. Similarly, Rps14 overexpression in the mice cochlea could promote supporting cells proliferation by activating the Wnt signalling pathway. In addition, over-expressing Rps14 induced hair cells regeneration in the organ of Corti, and lineage tracing showed that the new hair cells had transformed from Lgr5+ progenitors. In conclusion, our analysis reveals the potential role of Rps14 in driving hair cell regeneration in mammalian.

Prospect of acromegaly therapy: molecular mechanism of clinical drugs octreotide and paltusotine.

Somatostatin receptor 2 (SSTR2) is highly expressed in neuroendocrine tumors and represents as a therapeutic target. Several peptide analogs mimicking the endogenous ligand somatostatin are available for clinical use, but poor therapeutic effects occur in a subset of patients, which may be correlated with subtype selectivity or cell surface expression. Here, we clarify the signal bias profiles of the first-generation peptide drug octreotide and a new-generation small molecule paltusotine by evaluating their pharmacological characteristics. We then perform cryo-electron microscopy analysis of SSTR2-Gi complexes to determine how the drugs activate SSTR2 in a selective manner. In this work, we decipher the mechanism of ligand recognition, subtype selectivity and signal bias property of SSTR2 sensing octreotide and paltusotine, which may aid in designing therapeutic drugs with specific pharmacological profiles against neuroendocrine tumors.

Increased mitophagy protects cochlear hair cells from aminoglycoside-induced damage.

Aminoglycosides exhibit ototoxicity by damaging mitochondria, which in turn generate reactive oxygen species that induce hair cell death and subsequent hearing loss. It is well known that damaged mitochondria are degraded by mitophagy, an important mitochondrial quality control system that maintains mitochondrial homeostasis and ensures cell survival. However, it is unclear whether dysregulation of mitophagy contributes to aminoglycoside-induced hair cell injury. In the current study, we found that PINK1-PRKN-mediated mitophagy was impaired in neomycin-treated hair cells. Our data suggested that mitochondrial recruitment of PRKN and phagophore recognition of damaged mitochondria during mitophagy were blocked following neomycin treatment. In addition, the degradation of damaged mitochondria by lysosomes was significantly decreased as indicated by the mitophagic flux reporter mt-mKeima. Moreover, we demonstrated that neomycin disrupted mitophagy through transcriptional inhibition of Pink1 expression, the key initiator of mitophagy. Moreover, we found that neomycin impaired mitophagy by inducing ATF3 expression. Importantly, treatment with a mitophagy activator could rescue neomycin-treated hair cells by increasing mitophagy, indicating that genetic modulation or drug intervention in mitophagy may have therapeutic potential for aminoglycoside-induced hearing loss.Abbreviations: AAV: adeno-associated virus; ABR: auditory brainstem response; ATF3: activating transcription factor 3; ATOH1/MATH1: atonal bHLH transcription factor 1; BafA1: bafilomycin A1; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; COX4I1/COXIV: cytochrome c oxidase subunit 4I1; CTBP2/RIBEYE: C-terminal binding protein 2; DFP: deferiprone; EGFP: enhanced green fluorescent protein; FOXO3: forkhead box O3; GRIA2/GLUR2: glutamate receptor, ionotropic, AMPA2 (alpha 2); HC: hair cell; HSPD1/HSP60: heat shock protein 1 (chaperonin); IHC: inner hair cell; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MYO7A: myosin VIIA; OPTN: optineurin; OMM: outer mitochondrial membrane; PRKN: parkin RBR E3 ubiquitin protein ligase; PINK1: PTEN induced putative kinase 1; RT-qPCR: real-time quantitative polymerase chain reaction; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; TUNEL: Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling; USP30: ubiquitin specific peptidase 30; XBP1: X-box binding protein 1.

Dock4 is required for the maintenance of cochlear hair cells and hearing function.

Auditory hair cells (HCs) are the mechanosensory receptors of the cochlea, and HC loss or malfunction can result from genetic defects. Dock4, a member of the Dock180-related protein superfamily, is a guanine nucleotide exchange factor for Rac1, and previous reports have shown that Dock4 mutations are associated with autism spectrum disorder, myelodysplastic syndromes, and tumorigenesis. Here, we found that Dock4 is highly expressed in the cochlear HCs of mice. However, the role of Dock4 in the inner ear has not yet been investigated. Taking advantage of the piggyBac transposon system, Dock4 knockdown (KD) mice were established to explore the role of Dock4 in the cochlea. Compared to wild-type controls, Dock4 KD mice showed significant hearing impairment from postnatal day 60. Dock4 KD mice showed hair bundle deficits and increased oxidative stress, which eventually led to HC apoptosis, late-onset HC loss, and progressive hearing loss. Furthermore, molecular mechanism studies showed that Rac1/ß-catenin signaling was significantly downregulated in Dock4 KD cochleae and that this was the cause for the disorganized stereocilia and increased oxidative stress in HCs. Overall, our work demonstrates that the Dock4/Rac1/ß-catenin signaling pathway plays a critical role in the maintenance of auditory HCs and hearing function.

Tetrandrine Prevents Neomycin-Induced Ototoxicity by Promoting Steroid Biosynthesis.

Aminoglycoside antibiotics are widely used for the treatment of serious acute infections, life-threatening sepsis, and tuberculosis, but all aminoglycosides cause side effects, especially irreversible ototoxicity. The mechanisms underlying the ototoxicity of aminoglycosides need further investigation, and there are no effective drugs in the clinic. Here we showed that tetrandrine (TET), a bioactive bisbenzylisoquinoline alkaloid derived from Stephania tetrandra, ameliorated neomycin-induced cochlear hair cell injury. In both in vitro and in vivo experiments we found that TET administration significantly improved auditory function and reduced hair cell damage after neomycin exposure. In addition, we observed that TET could significantly decrease oxidative stress and apoptosis in hair cells after neomycin exposure. Finally, RNA-seq analysis suggested that TET protected against neomycin-induced ototoxicity mainly by promoting steroid biosynthesis. Collectively, our results provide pharmacological evidence showing that TET may be a promising agent in preventing aminoglycosides-induced ototoxicity.

AAV-ie-K558R mediated cochlear gene therapy and hair cell regeneration.

The cochlea consists of multiple types of cells, including hair cells, supporting cells and spiral ganglion neurons, and is responsible for converting mechanical forces into electric signals that enable hearing. Genetic and environmental factors can result in dysfunctions of cochlear and auditory systems. In recent years, gene therapy has emerged as a promising treatment in animal deafness models. One major challenge of the gene therapy for deafness is to effectively deliver genes to specific cells of cochleae. Here, we screened and identified an AAV-ie mutant, AAV-ie-K558R, that transduces hair cells and supporting cells in the cochleae of neonatal mice with high efficiency. AAV-ie-K558R is a safe vector with no obvious deficits in the hearing system. We found that AAV-ie-K558R can partially restore the hearing loss in Prestin KO mice and, importantly, deliver Atoh1 into cochlear supporting cells to generate hair cell-like cells. Our results demonstrate the clinical potential of AAV-ie-K558R for treating the hearing loss caused by hair cell death.