In vitro and in vivo models: What have we learnt about inner ear regeneration and treatment for hearing loss?

Abstract

The sensory cells of the inner ear, called hair cells, do not regenerate spontaneously and therefore, hair cell loss and subsequent hearing loss are permanent in humans. Conversely, functional hair cell regeneration can be observed in non-mammalian vertebrate species like birds and fish. Also, during postnatal development in mice, limited regenerative capacity and the potential to isolate stem cells were reported. Together, these findings spurred the interest of current research aiming to investigate the endogenous regenerative potential in mammals. In this review, we summarize current in vitro based approaches and briefly introduce different in vivo model organisms utilized to study hair cell regeneration. Furthermore, we present an overview of the findings that were made synergistically using both, the in vitro and in vivo based tools.

Introduction

Genetic and acquired hearing loss affects 1 in 5 people translating to approximately 1.57 billion globally (GBD-Hearing-Loss-Collaborators, 2021). Furthermore, global disease burden is expected to continually rise as life expectancy and societal noise burdens increase. For many patients with sensorineural hearing loss (SNHL) two options are available to overcome the limitations associated with the disease. The first option is a hearing aid, which is effective in supporting residual hearing. However, acceptance among patients is low and only approximately 20% of people with hearing loss use hearing aids (Larson et al., 2000). The second option is a cochlear implant for those with severe hearing loss, but outcomes are variable and cannot fully restore hearing. Both options are technical solutions to a biological problem and a medical treatment to cure hearing loss remains to be developed.

Hearing loss, specifically SNHL, is due to loss or damage of mechanosensory cells within the organ of Corti of the cochlea. The organ of Corti is constituted by two cell types, hair cells (HCs) and supporting cells (SCs). Upon HC loss, SCs remain and have been identified as the major target in HC regeneration. However, with the expression of the cell cycle inhibitor p27Kip1 (Chen and Segil, 1999; Löwenheim et al., 1999), SCs become postmitotic and are deemed nonregenerative in mammals (Corwin and Cotanche, 1988). Therefore, numerous efforts are underway to examine the mechanisms regulating inner ear specification, endogenous repair, and the potential of cell replacement therapies.

Research efforts have used various in vitro and in vivo models to study inner ear regeneration. In vivo based approaches have encountered challenges owing to the limited number of HCs available. In contrast, in vitro based approaches may be able to overcome the small cell numbers; however, efficient differentiation of SCs and HCs reaching adult-like stages has been challenging. Nevertheless, many signaling pathways and biological processes critical for inner ear specification, proliferation, and differentiation have been identified. Both in vitro and in vivo experiments have utilized various mammalian and nonmammalian species, such as zebrafish, chicken, and mice. In the first half of this review, we will provide an overview of the current in vitro and in vivo based approaches (Figure 1). In the second half of the review, we will summarize what we have learnt about HC regeneration, which is based on experiments synergistically utilizing in vitro and in vivo based approaches.

Figure 1. Approaches and Model Organisms in Regenerative Research of the Inner Ear.

Cell lines, 3D-organoids, and explants are in vitro approaches used to perform drug screening, disease modeling, and to explore stem cell therapies. In vivo model organisms like fish, chicken, and the mouse are used to study development and endogenous regeneration to develop new hypotheses and validate findings from in vitro based experiments.

In vitro Approaches

Scalability and the potential for high throughput applications are the two hallmarks of in vitro based experiments that allow for screening of drug libraries and small molecules. Additionally, available in vitro protocols have been translated to study ototoxicity and different aspects of regeneration, which will be discussed below.

Immortalized Cell Lines

Inner ear cell lines, like UB/OC-1/2 (Rivolta et al., 1998) or the HEI-OC1 (Kalinec et al., 2003), were first developed to provide screening tools for ototoxic reagents. To generate cell lines representing different levels of inner ear maturation, relevant tissues were isolated from the Immortomouse (Jat et al., 1991) at various developmental time points. Generally, cell culture conditions at 33°C with media supplemented with γ-interferon support stable proliferation, whereas increasing the temperature to 39°C and withdrawal of γ-interferon induces differentiation of the inner ear cell line. Differentiation of UB/OC-1/2 or HEI-OC1 cell lines does not generate specific inner ear cell types, like outer HCs or Deiters’ cells, but the transcriptomic profiles are reminiscent of inner ear tissue. Although differentiation (Carpena et al., 2021) has been studied with this model, the majority of studies examine ototoxicity (Cho et al., 2021; Dhukhwa et al., 2021; Gonçalves et al., 2019) and numerous other topics like apoptotic pathways, autophagy (Zhu et al. 2021), senescence (Zhu et al., 2021), and mechanisms of cell protection (Ingersoll et al., 2020; Kather et al., 2021; Wen et al., 2020).

In contrast, immortalized multipotent otic progenitor (iMOP) cells (Kwan et al., 2015) were generated by viral overexpression of C-MYC and supplemented with bFGF. iMOPs have been used to investigate self-renewal potential, differentiation of inner ear cell types (Azadeh et al., 2016), and the interplay between C-MYC and SOX2 (Kwan et al., 2015). In summary, inner ear cell lines provide valuable tools in inner ear research. However, the lack of resemblance of specific inner ear cell types upon differentiation, limits their use in regenerative research. Furthermore, stem cell-based approaches and directed differentiation of HCs have evolved as widely used alternatives.

Stem Cell-Based Approaches

A multitude of different stem cell types have been utilized to generate inner ear cell types. More recently, research has focused on tissue specific stem cells (Kubota et al., 2021; Oshima et al., 2007; White et al., 2006) and pluripotent stem cells (Koehler et al., 2013; Li et al., 2003b), while neuronal stem cells (Wei et al., 2008) and mesenchymal stem cells (Jeon et al., 2007) were explored to a lesser extent. Generally, compared to explant culture or in vivo applications, stem cell-based approaches hold the promise to provide larger quantities of HCs for analysis.

Tissue Specific Stem Cells

The majority of tissue specific stem cells are isolated from mouse inner ears (Diensthuber et al., 2014; Li et al., 2003a; McLean et al., 2016; Oshima et al., 2007; Rousset et al., 2020a) and less frequently from human organ of Corti, utricle, and spiral ganglion (Chen et al., 2009; Roccio et al., 2018; Senn et al., 2020). Sphere forming culture conditions allow for propagation of the cells, and differentiation occurs under adherent culture conditions combined with growth factor withdrawal or co-culture with feeder cells (Oshima et al., 2007; White et al., 2006). HC-like cells surrounded by SC-like cells can be created under these culture conditions. Initially, this approach suffered from low HC numbers, but later iterations of the protocol increased the HC yield through use of small molecules or flow sorting of defined cell populations (Kubota et al., 2021; McLean et al., 2017). Although the assay relies on primary tissue, which implies regular use of animals to generate new HC-like cells, several regenerative studies use this model. For example, Notch inhibitors and glycogen synthase kinase 3β (GSK3β) inhibitors were examined and shown to increase the potential to generate HCs from oto-spheres (McLean et al., 2017; Mizutari et al., 2013; Roccio et al., 2015). The sphere assay was also used to study the impact of reactive oxygen species on neurons (Rousset et al., 2020b) and DNA methylation at the Sox2 locus (Waldhaus et al., 2012).

Pluripotent Stem Cells

While the use of tissue specific stem cells has evolved over the past two decades, pluripotent stem cell-based protocols were developed in parallel. Early versions started with the induction of ectoderm and non-neuronal ectoderm and continued differentiation toward otic fates under adherent culture conditions (Chen et al., 2016; Chen et al., 2012; Ealy et al., 2016; Oshima et al., 2010; Ronaghi et al., 2014; Tang et al., 2016). Generally, those protocols resulted in the generation of HC-like cells or their respective progenitor cells in a 2D-culture environment. More recently, robust differentiation of HCs in 3D-organoids has been reported (Koehler et al., 2013). The aggregate based protocols allow for the study of HCs in a tissue-like context together with SCs and neurons (Chen et al., 2012; Jeong et al., 2018; Koehler et al., 2017; Matsuoka et al., 2017; Shi et al., 2007). Some of the major advantages of the pluripotent stem cell approaches are the reduced use of laboratory animals in comparison to all alternative approaches, and readily available protocols for mouse (Abboud et al., 2017; Koehler et al., 2013; Li et al., 2003b; Nie et al., 2017; Oshima et al., 2010; Ouji et al., 2012) and human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs) (Chen et al., 2012; Ding et al., 2016; Ealy et al., 2016; Gunewardene et al., 2014; Hosoya et al., 2017; Jeong et al., 2018; Koehler et al., 2017; Lahlou et al., 2018; Matsuoka et al., 2017; Mattei et al., 2019; Nie and Hashino, 2020; Ohnishi et al., 2015; Ronaghi et al., 2014; Shi et al., 2007). Specifically, human iPSCs aggregates were used to study deafness genes and mutations for genes like USH2A (Liu et al., 2021), MYO15A (Chen et al., 2016), MYO7A (Tang et al., 2016), and SLC26A4 (Chen et al., 2020). As methods advance, the use of human iPSCs will likely allow for the modeling of complex diseases, like the Waardenburg syndrome (Huang et al., 2021).

Current limitations of the pluripotent stem cell-based approaches relate to the cellular output of the current protocols. Several studies show that most HCs generated appear to be of vestibular phenotype (Jeong et al., 2018; Koehler et al., 2017; Mattei et al., 2019). However, with respect to modeling SNHL or age-related hearing loss, auditory HCs have yet to be generated. Therefore, protocol variations that could tip the balance from vestibular toward auditory HCs remain to be developed. Furthermore, HC-like cells generated in 3D-culture are encapsulated in vesicles that develop from the aggregate. This hinders the use of aggregates in high throughput imaging-based approaches as needed for drug development.

Direct Programming

As mentioned above, challenges with organoids are related to the encapsulation of the HCs. To overcome the need for the 3D-aggregates, HCs generated in a single epithelial layer would allow for automated imaging-based screens, especially when used in connection with fluorescent reporter expression (Costa et al., 2015; Menendez et al., 2020). Menendez et al., were able to directly program embryonic fibroblasts, adult tail tip fibroblasts, and postnatal murine SCs with a 4-transcription factor cocktail (SIX1, ATOH1, POU4f3, and GFI1) that reprograms target cells into HCs (Menendez et al., 2020). Furthermore, these induced HCs (iHCs) are a promising model that can be scaled up to identify ototoxic agents, factors significant for HC maturation, regeneration, and function.

Tissue & Organ Culture

Ex vivo organ culture models provide two major advantages compared to stem cell-based approaches. 1) The complex cellular architecture of the sensory epithelium is preserved and 2) cells of the explant represent a relatively homogenous maturation stage as compared to the developmental variability seen in stem cell-based approaches. Different protocols have been developed starting with the culture of embryonic (Haque et al., 2015) and postnatal sensory inner ear organs of mouse, human, and chicken (Landegger et al., 2017; Oesterle et al., 1993; Ogier et al., 2019). Specifically, a variation of the protocol was established to preserve the 3D-tissue context during culture and individual sensory organs were embedded and cultured in gels (Gnedeva et al., 2018). Alternatively, whole inner ear bony labyrinths (Arnold et al., 2010; Hahn et al., 2008) can be cultured as well. To create culture conditions that recapitulate in vivo conditions most accurately, the bony labyrinths were supported with buoyancy beads to generate a microgravity environment. Generally, ex vivo organ culture protocols allow for the culture of sensory epithelia for up to 7 days in vitro. However, a maximum culture duration of 14 days with surviving HCs has been reported (Nayagam et al., 2013; Sobkowicz et al., 1975). Due to its versatility, the assay was used in many studies covering ototoxicity and oto-protection (Ingersoll et al., 2020; Kopke et al., 1997; O’Sullivan et al., 2020; Perny et al., 2017; Tropitzsch et al., 2014; Tropitzsch et al., 2019). Furthermore, the effects of different inhibitors on cell differentiation and regeneration for LATS kinases (Kastan et al., 2021; Rudolf et al., 2020), p27Kip1 (Walters et al., 2014), GSK3β (Ellis et al., 2019; Roccio et al., 2015), and Notch pathway (Mizutari et al., 2013) were tested ex vivo.

Current protocols face two challenges for the future: 1) the limited duration of culture period for primary cultures and 2) the inability to culture adult inner ear tissues. Furthermore, the presence of resident macrophages from isolated tissues may interfere with experimental outcomes (Francis and Cunningham, 2017). However, the ability to overcome the limited number of progenitor cells and screen both ototoxic and regenerative compounds before moving in vivo are the largest advantage to in vitro experimentation.

In vivo Regeneration

Humans lack the capacity to regenerate lost HCs. In contrast, non-mammalian vertebrate species and neonatal mice show the capacity to regenerate lost HCs after trauma. While this is not an exhaustive list of model organisms employed, the following section will review some common vertebrate model organisms used to study HC regeneration in vivo.

Zebrafish

Zebrafish are a classic model system for developmental biology, molecular biology, regeneration, and with increasing frequency, drug screens (Koleilat et al., 2020; Ou et al., 2009; Teitz et al., 2018; Thomas et al., 2015). Just like in mammals, fish utilize mechanosensitive HCs in the inner ear for hearing (Pickett and Raible, 2019). Additionally, HCs can be found in the lateral line organ distributed along the body’s surface, and these HCs are turned over throughout the life cycle of the fish (Baxendale and Whitfield, 2016; Pickett and Raible, 2019). Following damage, lateral line HC regeneration occurs within 48 hours. Besides the direct accessibility of HCs in the lateral line, the major advantages of the zebrafish as a model organism for functional HC regeneration are owing to the large amount of offspring at a single time, optical clarity for imaging, and access to a vast number of genetic mutants. Numerous studies examining regeneration and ototoxicity have been performed owing to the ease in modeling genetic hearing loss and drug delivery through the water (Koleilat et al., 2020; Ou et al., 2009; Teitz et al., 2018; Thomas et al., 2015). Additionally, high throughput in vivo screening of otoprotective and ototoxic compounds is feasible in the zebrafish model system due to their small size and ability to fit into 96-well plates. Limitations faced when using zebrafish are the lack of different HC phenotypes, like inner and outer HCs, and the evolutionary distance to the mammalian counterparts. Notwithstanding, zebrafish studies have contributed to better understanding genetic and acquired hearing loss and regenerative mechanisms.

Chicken

Like zebrafish, birds have the capacity to regenerate lost HCs in the auditory and vestibular organs as well (Girod et al., 1991; Morest and Cotanche, 2004; Oesterle et al., 1993; Warchol and Corwin, 1996). Unlike in zebrafish, HCs are not constantly turned over throughout life and must first undergo damage in order to regenerate (Corwin and Cotanche, 1988; Girod et al., 1991). Chick embryos have classically been a strong model system for developmental biology owing to the ease of access to the embryo during development through shell windowing (Amprino and Camosso, 1959; Kieny, 1959; Waddington, 1950). Like the murine cochlea (Son et al., 2012), the avian basilar papilla is tonotopically organized (Girod et al., 1991; Son et al., 2015). More recent mechanistic studies of avian inner ear cells using single-cell sequencing during development and regeneration have helped to resolve much of the molecular basis of HC regeneration (Alvarado et al., 2011; Benkafadar et al., 2021; Janesick et al., 2021). Furthermore, comparative examination of mouse vs. chicken SCs after damage holds promise to highlight the differences in the cellular response between both species. The shorter evolutionary distance between birds and humans and the accessibility of the embryo in ovo has spurred additional interest in the chicken as model organism for HC regeneration (Alvarado et al., 2011; Benkafadar et al., 2021; Girod et al., 1991; Perl et al., 2018; Son et al., 2015).

Mouse

Mice were extensively studied as a mammalian model system for HC regeneration or the lack thereof. While neonatal mice show the ability to regenerate HCs (Chai et al., 2012; Cox et al., 2014; Oshima et al., 2007; Shi et al., 2012; White et al., 2006), the organ of Corti in adult mice does not regenerate spontaneously (Mizutari et al., 2013). Furthermore, mice exhibit age related hearing loss similarly to humans (Shone et al., 1991). Additional experimental advantages include the potential to isolate inner ear stem cells from neonatal mice as discussed earlier (Cox et al., 2014; Oshima et al., 2007; White et al., 2006). Furthermore, genetically modified mouse strains have paved the way to examine the molecular basis of human disease. For example, the shaker-1 mouse carries a mutation in MyoVIIa-gene and therefore served as a model for Usher syndrome (el-Amraoui et al., 1996; Gibson et al., 1995). Comparing the findings from different model organisms suggest that the molecular machinery necessary to regenerate HCs may be present in adult mammals; nevertheless, strategies to activate the regenerative potential remain to be determined.

What have we learnt about hair cell regeneration?

Our current knowledge about cell biological processes and the molecular pathways controlling regeneration is supported by various in vivo and in vitro based experiments. In the following section, findings will be summarized. Mechanosensory HCs are embedded in a layer of SCs. This conformation of cells is conserved across various model organisms from fish to humans. Both HCs and SCs share a common progenitor cell during cochlear development (Xu et al., 2017), and following damage the presence of SCs is required for HC regeneration (Bhatt et al., 2001; Izumikawa et al., 2008). HC regeneration occurring in fish, birds, and postnatal mammals relies on two cellular mechanisms (Figure 2):

Figure 2. Mechanisms of Hair Cell Regeneration.

HCs regenerate through a) transdifferentiation, where a SC directly converts into a HC or b) through mitotic regeneration, where SCs first proliferate, then the daughter cells differentiate into new HCs and/or SCs. Different molecular pathways involved were identified.

1) Transdifferentiation occurs when HC damage results in a direct conversion of SCs into HCs or 2) through mitotic regeneration when SCs re-enter the cell cycle, divide, and the daughter cells differentiate into new HCs as well as SCs. While HC regeneration at the cellular level has been studied for decades, the molecular mechanisms controlling the regenerative process are still not fully understood. Below, we discuss several signaling pathways, such as Wnt, Notch, Shh, and Hippo, which have been shown to be involved in HC development and regeneration.

Notch Signaling

Notch signaling within the organ of Corti was first examined for its contribution to prosensory domain specification and HC differentiation (Daudet and Lewis, 2005; Kiernan et al., 2001; Lanford et al., 1999; Maass et al., 2015; Woods et al., 2004). Specifically, the molecular mechanism controlling HC differentiation spurred the interest in potential regenerative applications. During HC differentiation, Notch singling functions through lateral inhibition; HCs express the Notch-ligand, that upon binding to its receptor inhibits SCs from differentiating into HCs. Mechanistic analysis has identified the bHLH transcription factor Atoh1 as a downstream target, which is negatively regulated by Notch signaling (Driver et al., 2013; Jones et al., 2006). Inhibition of Notch signaling was found to enhance existing regenerative capacity in avian (Alvarado et al., 2011), zebrafish (Jiang et al., 2014), and perinatal mice (Yamamoto et al., 2006) through trans-differentiation and mitotic regeneration. In contrast, neonatal HC differentiation was decreased in a murine conditional Notch overexpression model (McGovern et al., 2018). Related to these findings, delivery of γ-secretase inhibitor aiming to block Notch signaling after noise damage in the adult mouse ear was explored (Mizutari et al., 2013) and clinical trials for several γ-secretase inhibitors followed (Nakajima, 2015).

Wnt Signaling

Canonical Wnt signaling contributes to inner ear development controlling progenitor cell proliferation and HC differentiation (Bok et al., 2007a; Chai et al., 2012; Dabdoub et al., 2003; Jacques et al., 2012). Ectopic activation of Wnt signaling induces proliferation of prosensory cells and induces regeneration of HCs in perinatal mice (Chai et al., 2012; Song and Wang, 2020). Conversely, pharmacologic blocking of Wnt signaling or genetic targeting decreases proliferation in the prosensory domain. Lgr5 was identified as a downstream target of Wnt-signaling and successfully established as Wnt-reporter (Chai et al., 2011; Shi et al., 2012). Flow sorting of Lgr5+ cells allows for enrichment of a defined progenitor pool from the postnatal cochlea, which were found to possess the capacity of self-renewal and to differentiate into new HCs (Bramhall et al., 2014; Chai et al., 2012). These studies suggest that the Lgr5+ cells possess the capacity for regeneration. Like other tissues during development, the Wnt and Notch pathways intersect during tissue specification (Chen et al., 2002; Clevers, 2006; Johnson Chacko et al., 2020; Lanford et al., 1999). Specifically, both Wnt and Notch pathways regulate expression of key HC transcription factor, Atoh1, although in a converse manner (Chen et al., 2002; Jeon et al., 2011; Nakajima, 2015). This emphasizes the need for precise temporal control of signaling pathway activity for correct specification and differentiation.

Sonic Hedgehog Signaling

Sonic hedgehog (Shh) signaling plays an important role in patterning the otocyst and, together with Wnt signaling, defines the dorsal ventral axis of the developing cochlea. (Bok et al., 2005; Liu et al., 2002). Shh knock out mice demonstrate that Shh signaling is not required for otic vesicle formation but required for induction and specification of the sensory organs (Riccomagno et al., 2002). Additionally, Shh has roles in regulating the tonotopic patterning of cells along the longitudinal axis of the cochlea (Son et al., 2015) and in supporting the undifferentiated prosensory progenitor state in the cochlear apex (Bok et al., 2007b; Bok et al., 2013; Liu et al., 2010; Tateya et al., 2013). In neonatal mice following HC damage, overexpression of Shh induces SC proliferation and HC regeneration (Chen et al., 2017). Additionally, the newly formed HCs were derived from Lgr5+ progenitors, which further emphasizes the interconnectivity of signaling pathways during cochlear development and regeneration.

Hippo Signaling

Hippo signaling through YAP/TEAD transcriptional regulators is a conserved regulatory pathway in numerous tissues and across species (Calses et al., 2019; Dey et al., 2020; Galli et al., 2015; Gnedeva et al., 2020; Rudolf et al., 2020; Zanconato et al., 2015). Although the transcriptional targets of YAP/TEAD are largely cell type specific, similar functions in controlling proliferation, apoptosis, tissue regeneration, and organ size have been observed (Galli et al., 2015; Gnedeva et al., 2020; Zanconato et al., 2015). Recently, studies have highlighted the role of Hippo signaling in both murine and avian inner ear size (Gnedeva et al., 2020; Rudolf et al., 2020). In the developing murine organ of Corti, TEAD target gene expression was observed to decrease sharply as the organ of Corti exit cell cycle around E14.5 (Gnedeva et al., 2020). Additionally, conditional knock-out of YAP reduces the size of the utricle and organ of Corti at E18.5. Within the adult avian utricle, nuclear YAP protein and proliferation were observed, while sequestered YAP was found in adult murine utricular SCs (Rudolf et al., 2020). Additionally, conditional overexpression of YAP in mice at P10 resulted in cell cycle reentry following damage (Gnedeva et al., 2020). Recently, the small molecule TRULI was identified as a compound that inhibits Lats kinases (LATS1/2), which are responsible for YAP cytoplasmic sequestration and inactivation (Kastan et al., 2021). Adult mice treated with TRULI exhibited proliferation in utricle SCs but not the organ of Corti. These studies emphasize the importance of Hippo signaling in inner ear proliferation, organ size regulation, and as a potential target for HC regeneration.

Strategies in Treatment of Hearing Loss

Cell Replacement Therapy

Cell replacement therapy is an area of research located at the intersection of in vitro and in vivo based experimentation. The success in differentiating stem cells into inner ear cell types raised the interest in regeneration of lost HCs or spiral ganglion neurons by transplanting undifferentiated stem cells or partly differentiated stem cells into damaged ears. However, studies in mice have faced challenges since the high potassium concentration in the endolymph poses a challenge for stem cell survival (Hu et al., 2004). When mESCs were transplanted into undamaged rat cochlea only about 1% of the stem cells survived and integrated. Improvements were made by transplanting stem cells into damaged ears, which resulted in 25% of the cells surviving for up to 4 weeks (Regala et al., 2005). Further progress was made with supplementation with BDNF and chondroitinase ABC enzyme (ChABC) to improve survival and migration, respectively (Palmgren et al., 2012). Further challenges are being addressed regarding differentiation into functional HCs or neurons (Gokcan et al., 2016). Generally, injection of undifferentiated ESCs is associated with the formation of teratomas (Chen et al., 2018; Fukuda et al., 2006). However, the use of differentiated stem cells is intended to mitigate both problems. Differentiation of neural cell types that are subsequently implanted with outgrowing neurites resulted in improved auditory brainstem responses (Chen et al., 2012; Fukuda et al., 2006; Ishikawa et al., 2017; Lopez-Juarez et al., 2019). In summary, functional recovery of spiral ganglion neurons or HCs by replacement remains a promising strategy but outcomes need to be improved.

Gene Therapy

Gene therapy including, gene transfer and gene editing, holds promise in treating both genetic and acquired deafness. Recent years have seen immense progress in developing methodologies for gene delivery to the cochlea to induce regeneration. In the following section we will briefly summarize some advances in gene therapy in mammals and highlight studies using Atoh1 gene delivery.

Studies have used in vivo viral delivery of genes, such as neurotropic factor-9, TGF-beta 1, and GDNF, following ototoxic damage in rats and guinea pigs (Kawamoto et al., 2003; Zheng et al., 2011; Zheng et al., 2013). Additionally, mice with genetic mutations in Tmc1, a gene associated with genetic deafness, were able to largely recover hearing when treated with Tmc1 in an adeno-associated virus (AAV) based approach (Nist-Lund et al., 2019; Valentini et al., 2020; Wu et al., 2021). Delivery of reagents through the round window of the cochlea allows for a more directed delivery of gene therapy in vivo (Ivanchenko et al., 2021; Valentini et al., 2020). Mice with Usher syndrome treated with AAVs delivering Clarin-1 (Dulon et al., 2018) or sans (Emptoz et al., 2017) through the intracochlear route both display some preservation of hearing. Variations of AAVs have been developed in order to increase the safety and efficacy for human treatment (Valentini et al., 2020). Additionally, nonviral approaches are being developed using liposomes, peptides, and polymers in order to deliver genes for expression or for genetic alteration with CRISPR/CAS9 (Dong et al., 2019; Farooq et al., 2020). Specific mutations associated with hearing loss have been successfully corrected in stem cells derived from patients with genetic hearing loss (Chen et al., 2016; Dong et al., 2019; Farooq et al., 2020; Liu et al., 2021; Omichi et al., 2019). CRISPR/CAS9 correction of gene mutations could address the challenges faced with multigenic conditions and some hereditary mutations have the potential to be corrected before implantations of a blastocyst or in utero. However, the efficacy and specificity must first be improved before it is employed for treatment of humans.

While numerous genes are candidates for gene therapy, we will discuss one gene of importance, ATOH1. Atonal homolog 1 (ATOH1) is a transcription factor expressed in the early progenitors of the Organ of Corti and has been described as the master regulator of HC fate (Woods et al., 2004). ATOH1 is essential for HC differentiation and is regulated by Wnt and NOTCH signaling, as described above (Bai et al., 2021; Driver et al., 2013; Ellis et al., 2019; Jeon et al., 2011; Woods et al., 2004). Deafness causing mutations for the human ATOH1 gene have not been reported; however, due to its role as master regulator in hair cell development, initial gene therapeutic approaches focused on the transcription factor. Studies in mice have found that in utero gene delivery of Atoh1 resulted in excessive HCs within the cochlea (Gubbels et al., 2008). Furthermore, these excess HCs were functional in the postnatal animal. In both genetically deaf mice and those with HC damage from ototoxic drugs, Atoh1 gene delivery was able to induce HC regeneration (Izumikawa et al., 2008; Izumikawa et al., 2005; Staecker et al., 2007). These studies demonstrate the importance of Atoh1 in HC differentiation and regeneration. Further examination of Atoh1 transcriptome and interactome may provide more targeted therapies and open the possibilities for pathway specific small molecules.

While gene therapy isn’t available for human patients with genetic and acquired hearing loss at this time, research is steadily moving forward. Gene therapy has historically faced the challenge of viral vector safety, specific cell type delivery, and off target effects. Overcoming these challenges will be key in developing effective and safe clinical treatments.

Conclusions and Future Directions

A variety of protocols and methods have been established to overcome experimental limitations like encapsulation and sparsity of the inner ear cell types. Various in vivo and in vitro approaches were combined to identify multiple pathways enhancing the regenerative potential of the inner ear in perinatal mice. However, regeneration of HCs in the adult mammalian cochlea resulting in restored function remains elusive. To date, molecular mechanisms resulting in the loss of the regenerative potential, specifically in the mammalian cochlea, are still under investigation. Similarly, the translational potential related to differentiation of stem cell aggregates being utilized in personalized medicine or cell replacement therapies remains to be determined.