# Gene therapy for hearing loss: Current status and future prospects of non-viral vector delivery systems

Jean-Christophe Leclère, Remi Marianowski, Tristan Montier

► To cite this version:

Jean-Christophe Leclère, Remi Marianowski, Tristan Montier. Gene therapy for hearing loss: Current status and future prospects of non-viral vector delivery systems. Hearing Research, 2024, 453, pp.109130. 10.1016/j.heares.2024.109130. hal-05054222

HAL Id: hal-05054222

https://hal.science/hal-05054222v1

Submitted on 2 May 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
Review Article

# Gene therapy for hearing loss: Current status and future prospects of non-viral vector delivery systems

Jean-Christophe Leclère<sup>a,b,*</sup>, Remi Marianowski<sup>a,b</sup>, Tristan Montier<sup>c,d,*</sup>

<figure><img src="image_2.png" /></figure>

ARTICLE INFO

Keywords:

Non-viral vector

Gene therapy

Hearing loss

Congenital

Cationic lipids

Polymeric nanoparticles

Electroporation

ABSTRACT

Current therapeutic options for hearing loss rely on hearing aids, ossiculoplasty or cochlear implants. These devices have limitations, particularly in noisy acoustic environments. Therefore, interest in exploring aetiological treatments to improve not only auditory perception but also the quality of life of those affected is increasing. Gene therapy is a promising aetiological treatment that can fully restore auditory function. The success of gene therapy relies on the efficient delivery of therapeutic genes or genetic modifications to the cells of the inner ear that are designed to repair or replace defective genes and restore normal hearing function. Two main strategies for gene therapy involve the use of recombinant viral vectors and nonviral delivery vehicles.

Owing to their excellent diffusion properties and compatibility with sensory cells, recombinant viral vectors, particularly adeno-associated viruses (AAVs), have dominated gene therapy in the cochlea. However, recombi-
nant viral vectors have several drawbacks, such as limited transgene size, immunogenicity (particularly in ne-
onates), and potential need for repeat administration.

Nonviral vectors, such as cationic lipids and polymeric nanoparticles, are potential attractive alternatives. Nonviral vectors have several advantages, including low immunogenicity and unlimited transgene size. Recent studies have demonstrated significant auditory recovery in vivo using nonviral vectors in murine models. However, nonviral vectors are not as efficient as viral vectors in transferring genetic material.

An alternative to nanoparticles is the use of other methods, such as electroporation. The main advantage of electroporation is that it can be used in combination with cochlear implantation and can target surface cells, but this method has a risk of cell damage.

The goal of this review is to provide valuable insights into the current state of research on nonviral vectors for inner ear gene therapy and propose the exploration of innovative and effective gene therapy strategies for the treatment of hearing loss.

# 1. Introduction

Worldwide, nearly 20 % of the global population, >1.5 billion peo-
ple, live with hearing loss (World report on hearing, 2019). In developed
countries, approximately 1 in 500 newborns have severe to profound
hearing loss caused by genetic factors (Smith et al., 2005), with
approximately 50–60 % of all cases of hearing loss in children being due

to genetic factors. The prevalence of genetic hearing loss is greater in
certain populations, such as those with a history of consanguinity and in
some ethnic groups. There are over 400 genetic syndromes associated
with hearing loss, and over 100 genes have been identified that cause
nonsyndromic hearing loss.

Current treatments consist of two types of compensatory systems as
follows: hearing aids, which amplify sound; and cochlear implants,

Abbreviations: ABR, auditory brainstem response; AAV, adeno-associated viruses; BDNF, brain-derived neurotrophic factor; CMAP dendriplex, Na-carboxymethyl-beta-cyclodextrin-modified activated polyamidoamine dendrimers; HPNPs, Hyperbranched polylysine nanoparticles; IHC, Inner Hair Cell; LNPs, Lipid-based NanoParticles; OHC, Outer Hair Cell; PEI, Polyethyleneimine; PHEA, polyhydroxyethyl aspartamide; PLGA, Polylactic-co-glycolic acid; RWM, round window membrane.

<footnote><sup>a</sup> CHU de Brest, Service d'ORL et chirurgie cervico-faciale, 29200 Brest, France</footnote>

<footnote><sup>b</sup> Univ Brest, LIEN, 29200 Brest, France</footnote>

<footnote><sup>c</sup> Univ Brest, INSERM, EFS, UMR 1078 GGB-GTCA team, 29200 Brest, France</footnote>

<footnote><sup>d</sup> CHU de Brest, Service de Génétique Médicale et Biologie de la Reproduction, Centre de Référence des Maladies Rares "Maladies Neuromusculaires", 29200 Brest, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: leclere.jean-christophe@hotmail.fr, Jean-christophe.leclere@chu-brest.fr (J.-C. Leclère), tristan.montier@univ-brest.fr (T. Montier).</footnote>
which encode sound as electrical impulses that are delivered to the auditory nerve through an array of electrodes (Purcell et al., 2021). Although these devices usually help children with the development of speech and communication, they have certain limitations in terms of speech intelligibility in noise (Müller and Barr-Gillespie, 2015). These devices are currently unable to restore hearing to a completely normal state; hence, there is increasing interest in developing aetiologic treatments to improve hearing understanding and quality of life (Géléoc and Holt, 2014).

Gene therapies are being developed with the aim of fully restoring hearing and achieving better results than the current systems provide or to be used in parallel with other treatment modalities. While anatomical isolation of the cochlea may be an advantage for avoiding the proliferation of therapies, limited surgical access presents challenges, and there is a risk of worsening hearing loss with intervention. Various options to limit this risk are being investigated (Lee et al., 2020). For example, a therapeutic gene can be delivered through cochleostomy to cells in the inner ear to replace or repair a defective or missing gene, thereby restoring normal inner ear function (Delmaghani and El-Amraoui, 2020). As both nucleic acids and cell membranes are negatively charged, this helps prevent nucleic acids from spontaneously crossing the plasma membrane.

Several types of transgenes are used in gene therapy, including mRNAs, siRNAs, oligonucleotides, and plasmids. mRNA, a type of RNA molecule, carries genetic information to the ribosome, where it acts as a blueprint for protein synthesis. Short interfering RNAs (siRNAs) are double-stranded RNA molecules that selectively inhibit the expression of specific genes by targeting complementary mRNA molecules for degradation. Oligonucleotides are short sequences of nucleotides (RNA or DNA) that typically consist of 20-30 base pairs, and they modulate gene expression, repair genetic mutations, or inhibit protein synthesis. A plasmid is a small circular DNA molecule that replicates independently and is often used in target cells for long-term gene expression.

The successful introduction of genetic material into cells requires efficient and safe vectors. The vector must be able to compact nucleic acids and protect them throughout their journey from the point of administration to the nucleus. An ideal vector should be specific for the target tissue, nonimmunogenic, generate as few side effects as possible, and induce significant and functional expression of therapeutic transgenes. There are two main approaches for gene delivery as follows: recombinant viral vectors, which exploit the natural ability of viruses to penetrate host cells; and nonviral approaches, which are based on synthetic compounds and can be further divided into chemical and other approaches.

Currently, recombinant viral vectors, particularly adeno-associated viruses (AAVs), constitute the mainstay of gene therapy because of their excellent diffusion within the cochlea and inner hair cells. For example, AAV2.7m8 has excellent infection efficiency in cochlear hair cells, reaching 84.1% in inner hair cells (IHCs), 83.1% in outer hair cells (OHCs), and 86.1% in inner pillar cells (Isgrig et al., 2019). Injection of AAVs into the cochlea preserves hair cell morphology and orientation, with no significant differences in hair cell counts or auditory brainstem response thresholds between injected and uninjected ears, indicating minimal to no toxic effects of AAVs on hearing (Tan et al., 2019). A weakness of AAVs is the size of the transgene they can carry (4.7 kb). The use of dual AAVs has made it possible to overcome this size limitation to a certain extent and is being used for the otoferlin gene (Ghosh et al., 2011; Akil et al., 2019; Al-Moyed et al., 2019). However, the problem remains for larger genes. Another weakness is immunogenicity, especially because the target population is likely to be neonates (Kho, 2000). Currently, there is limited information on how the human inner ear responds to gene delivery by AAVs (Ishibashi et al., 2023). The use of an AAV as a vector to treat spinal muscular atrophy, a life-threatening disease, has been a major breakthrough. However, it is important to be aware of its potential side effects, particularly acute liver dysfunction, which has been observed in some young patients (Philippidis, 2022).

However, ocular gene therapy with AAV is associated with only an 8% cumulative incidence of serious adverse events, most of which are likely to be caused by the surgical procedure used to deliver the therapeutic vector (Sobh et al., 2023). Although the methods of delivery and sub-types of AAVs may vary, the potential for serious side effects associated with these vectors raises ethical concerns for its use in the treatment of hearing loss in children, particularly in view of the effectiveness of current functional treatments, without serious side effects. This treatment has recently been introduced in 6 children (Lv et al., 2024), leading to an improvement in speech perception owing to its efficacy; however, 48 adverse events were reported in these 6 patients, including 2 grade 3 events with uncertain aetiology.

Synthetic vectors, such as cationic lipids or polymeric nanoparticles, offer many advantages over viral vectors, particularly low immunogenicity (Scheper et al., 2009). Notably, siRNA molecules alone can diffuse from the middle ear to the inner ear across the round window membrane (RWM), and they are capable of inducing a silencing response to gene expression without evidence of histopathology in the sensitive inner ear. (Mukherjea et al., 2010).

Compared with viral vectors, synthetic vectors have many advantages, such as well-controlled production, high stability, low immunogenicity, and low production costs, and the size of the transgene they can carry is theoretically unlimited (Fig. 1). However, the main limitation of synthetic vectors is their ability to transfer genetic material, which is still lower than that of viral vectors. The efficiency of synthetic vectors varies depending on intrinsic factors, such as the chemical structure of the vector, the charge ratio for lipids or polymers, and the formulation characteristics of the associated auxiliary lipid (Le Corre et al., 2014; Belmadi et al., 2015). Extrinsic factors also influence efficiency during the gene delivery process; they depend on the delivery route and include cellular barriers and the extracellular environment.

Another option for gene transfer without the use of viral vectors is electroporation. This technique uses high-intensity electrical pulses to disrupt and permeabilize the cell membrane, allowing nucleic acids to enter (Mehier-Humbert and Guy, 2005). Electroporation is highly efficient and allows for precise control over the site of gene transfer; however, its efficiency is primarily applicable to surface cells, and the electrical pulses can potentially cause cell damage. Because this technique requires the insertion of electrodes, it can currently be used in conjunction with cochlear implantation to treat severe to profound deafness, as no new intervention is necessary.

The aim of this review is to provide an overview of the current state of research on nonviral vectors for inner ear gene therapy. Specifically, this review explores various types of nonviral vectors, including lipid-based nanoparticles, polymeric nanoparticles, and other methods. The advantages and limitations of each technique type are discussed, along with proposals for future studies.

## 1.1. Lipid-based nanoparticles (LNPs)

Lipid-based nanoparticles (LNPs) consist of a hydrophobic core and a hydrophilic outer layer, often forming a lipid bilayer structure or vesicle. By encapsulating genetic material, LNPs facilitate specific targeting by diffusion. One of the advantages of LNPs is that they can be reused without side effects (Lindberg et al., 2015). Currently, LNPs are under investigation for use as gene therapy vectors in various clinical settings. In 2018, patisiran [Onpattro] became the first drug using LNPs to be approved by the FDA (Suhr et al., 2015). Patisiran encapsulates siRNAs within an LNP for targeted delivery to the liver, where it modulates or inhibits the production of disease-causing proteins in hereditary thyretin amyloidosis, an autosomal dominant inherited disorder. More recently, LNPs have been used in mRNA vaccines against COVID-19 (Wang et al., 2021). The successful use of these vaccines on a large scale (hundreds of millions of people worldwide), with generally limited side effects (Li et al., 2022), has naturally increased interest in their development (Klein et al., 2021).
<table><caption>Fig. 1. Gene delivery methods for cochlear applications, highlighting their efficiency, advantages, and associated risks.</caption><thead><tr><th>Vector</th><th>Efficiency</th><th>Advantage</th><th>Disadvantage</th><th>Delivery</th></tr></thead><tbody><tr><td>Viral</td><td>Very High</td><td>Excellent diffusion<br>Can target all cell types<br>Best efficiency on IHC</td><td>Limited Size – 4.8 kb<br>Side effects (immunogenicity and re administration)<br>Risk of integration of plasmid sequence into host genome</td><td>Cochleostomy<br>RWM</td></tr><tr><td>Lipid-based NP</td><td>Low to High</td><td>No size limit (compatible with genome editing technologies)<br>Low immunogenicity<br>Can be reused</td><td>Uncertain long-term effects<br>Variable efficiency<br>OHC toxicity</td><td>Cochleostomy<br>RWM</td></tr><tr><td>Polymeric NP</td><td>Middle to High</td><td>Can cross RWM with encapsulated compound<br>Easy polymer modification</td><td>Side effects<br>ABR deterioration (PEI)<br>DPOAE deterioration (PHEA)</td><td>Cochleostomy<br>RWM</td></tr><tr><td>Electroporation</td><td>Low to high</td><td>Can be used in combination with cochlear implant<br>Good efficiency on the cochlea base<br>Possible in utero use</td><td>Risk of tissue damage risk<br>Limited efficiency on distant cells</td><td>Cochlear implant</td></tr><tr><td>Inorganic NP</td><td>Unknown</td><td>Good control of gene transfer area (gene gun)<br>Can target every cell (gene gun)<br>Can be used in combination with cochlear implant (silica)</td><td>No in vivo results<br>Tissue damage risk<br>Nonspecific cell targeting</td><td></td></tr></tbody></table>

## 1.2. Cationic lipid formulations

Cationic lipids, ranging from 80 to 120 nanometres in diameter, are composed of a positively charged (cationic) hydrophilic head and a hydrophobic tail. The positively charged portion enables both the formation of lipid-DNA-RNA complexes owing to the negative charge of nucleic acids and interaction with negatively charged cell membranes. The cationic lipid head groups can contain tertiary amines, allowing them to transform from neutral to positively charged particles under acidic pH conditions. The hydrophobic part promotes fusion with the cell membrane and the release of DNA/RNA into target cells (Afonso et al., 2016). Cationic lipids form liposomes or lipoplexes with DNA, which appear as spherical vesicles composed of a double layer of phospholipids enclosing an aqueous environment. Synthetic cationic lipids used for transfection can be coupled with auxiliary lipids to increase the destabilisation of the endosome after endocytosis, resulting in the effective release of DNA into the cytoplasm (Wrobel and Collins, 1995; Billiet et al., 2012).

Cationic lipids have been used in this type of application for approximately two decades (Wareing et al., 1999; Zheng and Gao, 2000; Belmadi et al., 2015) (Table 1).

Cationic lipids have potential use in adult animals. Several routes of administration have been tested, including cochleostomy and application to a gelatine sponge in contact with the round window membrane (Jero et al., 2001b; Zou et al., 2008). The peak efficacy has been reported to be approximately 3 days, depending on the promotor, but few long-term analyses have been performed. All tested cell types could be transfected, with varying efficiencies, but up to 39 % transfection could be achieved (Jero et al., 2001a). Auditory tests are not affected by surgery or these compounds, except when they are used at high concentrations (Staecker et al., 2001; Maeda et al., 2007). The use of

commercially available agents, such as Lipofectamine (Invitrogen, Ghent, Belgium), which are easy to use, has greatly increased access to this type of vector. Although the efficacy results vary widely (3 to 91 % transfection), this type of cationic lipid has become the most widely used (Zhang et al., 2011; Zuris et al., 2015).

One of the advantages of using cationic lipids lies in the potential for genome editing technologies, with a goal to directly modify the genetic mutation responsible for deafness to restore hearing. One promising approach involves the delivery of Cre recombinase and Cas9:sgRNA complexes to the organ of Corti via commercial vectors, such as RNAi-MAX and Lipofectamine 2000. Zuris et al. (Zuris et al., 2015) applied these vectors to mice between P0 and P2, with a specific focus on targeting outer hair cells, and they reported a recombination efficiency of 33 % with RNAiMAX and 91 % with Lipofectamine 2000. It is important to note that Lipofectamine 2000 induced toxicity in outer hair cells, but Zuris et al. did not quantify the extent of this toxicity. The limitations of this study include not evaluating the impacts of the intervention on hearing or effects on distant cells.

Genome modification to restore hearing or reduce hypoacusis in patients with progressive deafness is theoretically easier to achieve in autosomal dominant disorders. Building upon this premise, Gao et al. targeted Beethoven, a mouse model of dominant progressive hearing loss (Vreugde et al., 2002; Gao et al., 2018), by administering cationic lipid-encapsulated Cas9-gRNA complexes targeting the mutant allele transmembrane channel-like 1 (Tmc1) via cochleostomy to neonatal mice (P0-P2). This intervention resulted in a significant reduction in progressive hearing loss and an increase in hair cell survival. Additionally, there was a decrease in auditory brainstem response (ABR) thresholds in the injected ears compared with those in the ears that remained untreated or received injections of complexes targeting another gene. Compared with their uninjected counterparts, the acoustic
<caption>**Table 1**</caption>

Summary of cationic lipid-mediated transfection methods and results in inner ear cells (Ref.: reference, Effic: efficiency, ABR: auditory brainstem response, Tox.: toxicity, RWM: round window membrane, OHC: outer hair cell, IHC: inner hair cell, SGN: spiral ganglion neuron).

<table><thead><tr><th>Ref.</th><th>Vector</th><th>Trans-gene</th><th>Animal</th><th>Transf-lection age</th><th>Delivery</th><th>Analysis delay (days)</th><th>Targeted cells</th><th>Effic.</th><th>ABR</th><th>Tox.</th></tr></thead><tbody><tr><td>(Wareing et al., 1999)</td><td>Liposome (DDAB)</td><td>β-gal</td><td>guinea pig</td><td>adult</td><td>RWM, cochleostomy ± minipump</td><td>1,3,7,14</td><td>spiral limbus, spiral ligament, Reissner's membrane, spiral ganglion neurons</td><td>-</td><td>-</td><td>-</td></tr><tr><td>(Zheng and Gao, 2000)</td><td>Lipofectamine</td><td>Math1-eGFP</td><td>Rat</td><td>P3</td><td>Utricles</td><td>6-8</td><td>Hair cells, Supporting cells</td><td>-</td><td>-</td><td>-</td></tr><tr><td>(Jero et al., 2001a)</td><td>Liposome (POPC/CHIM/DDAB)</td><td>eGFP</td><td>Mouse</td><td>adult</td><td>gelatin sponge - RWM</td><td>3</td><td>Spiral limbus, spiral ligament, organ of corti, Reissner's membrane, spiral ganglion cells</td><td>39 %</td><td>-</td><td>-</td></tr><tr><td>(Jero et al., 2001b)</td><td>Liposome (POPC/CHIM/DDAB)</td><td>eGFP</td><td>Mouse</td><td>adult</td><td>gelatin sponge - RWM / cochleostomy</td><td>3</td><td>Spiral limbus, spiral ganglion cells, Reissner's membrane</td><td>-</td><td>-</td><td>-</td></tr><tr><td>(Staecker et al., 2001)</td><td>Liposome (DOTMA)</td><td>eGFP</td><td>Mouse</td><td>P90</td><td>cochleostomy</td><td>3</td><td>Spiral ganglion neurons, stria vascularis, supporting cells, mesenchymal cells</td><td>47 % (SGN)</td><td>-</td><td>Yes</td></tr><tr><td>(Maeda et al., 2007)</td><td>Liposome (DOTAP)</td><td>GJB2-eGFP</td><td>Mouse</td><td>adult</td><td>cochleostomy - RWM</td><td>3,5,12</td><td>Claudius cells, pillar cells, OHC, spiral limbus, spiral ligament</td><td>-</td><td>Normal</td><td>-</td></tr><tr><td>(Zou et al., 2008)</td><td>Liposome (Labrafac/Lipoid/Solutol)</td><td>NA</td><td>Rat</td><td>adult</td><td>gelatin sponge - RWM</td><td>0-7</td><td>Spiral ganglion cells, IHC, OHC pillar cells, spiral ligament, stria vascularis, Fibrocytes, spiral ganglion neuron, hair cell like cells</td><td>-</td><td>-</td><td>-</td></tr><tr><td>(Zhang et al., 2011)</td><td>Lipofectamine 2000</td><td>Math1-eGFP</td><td>Rat</td><td>P1-P5, adult</td><td>organ of corti explants</td><td>-</td><td></td><td>3 %</td><td>-</td><td>-</td></tr><tr><td>(Zuris et al., 2015)</td><td>Lipofectamine 2000</td><td>Cre</td><td>loxP-Tomato mice</td><td>P0</td><td>cochleostomy</td><td>5</td><td>OHC</td><td>91 %</td><td>-</td><td>Yes</td></tr><tr><td></td><td>Lipofectamine 2000</td><td>Cas9: sgRNA</td><td>Atoh1-GFP mice</td><td>P2</td><td>cochleostomy</td><td>5-10</td><td>OHC</td><td>13-20 %</td><td>-</td><td>No</td></tr><tr><td></td><td>Lipofectamine RNAiMAX</td><td>Cre</td><td>loxP-tdTomato mice</td><td>P0</td><td>cochleostomy</td><td>5</td><td>OHC</td><td>33 %</td><td>-</td><td>No</td></tr><tr><td>(Gao et al., 2018)</td><td>Lipofectamine 2001</td><td>Cas9: sgRNA</td><td>Mouse</td><td>P0-2, P40</td><td>cochleostomy (base, middle and apex-middle turn)</td><td>14</td><td>IHC</td><td>21 %</td><td>Better</td><td>-</td></tr><tr><td>(Tao et al., 2023)</td><td>Lipofectamine 2000</td><td>Cas9: sgRNA</td><td>Mouse</td><td>P0-2</td><td>cochleostomy (base, middle and apex-middle turn)</td><td>28-56</td><td>IHC</td><td>21 %</td><td>Better</td><td>-</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>OHC</td><td></td><td>OHC</td><td>28 %</td><td>Better</td><td>-</td></tr></tbody></table>

reflex responses of the injected Tmc1-mutated mice were enhanced.

Recently, the same group published an article on a mouse model carrying a dominant hearing loss mutation affecting the OHC, known as Oblivion (Tao et al., 2023). In vivo delivery of CRISPR-Cas9 ribonucleoprotein complexes using Lipofectamine 2000 at an early age specifically edited the Obl allele in this mouse model. Genome editing in vivo resulted in survival and functional restoration of outer hair cells, leading to partial recovery of hearing (average ABR threshold of 26 dB). Further, the most significant improvement was observed at frequencies below 32 kHz. However, these results were not sustained over time and declined significantly after 16 weeks.

In a double mutant mouse model with both Tmc1 and Oblivion mutations, causing digenic genetic hearing loss, delivery of Cas9/sgRNA targeting both mutations slightly improved hearing (average ABR threshold of 10 dB). While these gains are promising, especially considering the technical complexity of the correction, they need to be further improved to be of clinical significance. These results suggest that liposome-mediated delivery of these RNP-CRISPR complexes may be a potential strategy for restoring hearing in cases of dominant mutations in outer hair cell genes and digenic mutations in auditory hair cells.

Finally, a hybrid technique, using Lipofectamine 2000 as a vector for adeno-associated virus (AAV), was tested (Casey et al., 2020). Compared with the virus alone, this approach did not improve the transfection efficiency. Hence, the relevance of the immune response remains to be evaluated.

### 1.3. Other lipids

Another type of lipid used in the inner ear is neutral lipids (Miwa et al., 2021). Neutral lipids carrying brain-derived neurotrophic factor (BDNF) mRNA reduced the degeneration of spiral ganglion neurons in adult guinea pigs. Neutral lipids have been applied to the round window via a gelatine sponge, resulting in a greater number of spiral ganglion neurons at Day 14 in the lipid nanoparticle-treated group than in the saline control group, despite an overall decrease in the total number of spiral ganglion neurons. In addition, although no toxicity was observed in inner hair cells (IHCs), significant toxicity was observed in outer hair cells (OHCs), resulting in severe hearing loss.

### 1.4. Polymeric nanoparticles

As a more recent development in inner ear gene therapy, polymer nanoparticles (Tan et al., 2008; Zhang et al., 2011). They can cross membranes, such as the round window membrane. Polymers have the advantage of allowing a wide range of modifications, including the design of block and comb polymers. However, their elimination or biodegradation can potentially lead to toxic side effects, which may limit their use in clinical practice (Li et al., 2015). In this context, we review
the key findings of studies on this topic, focusing on polyactic-co-glycolic acid (PLGA), polyhydroxyethyl aspartamide (PHEA), polyethyleneimine (PEI), and hyperbranched polylysine nano-particles (HPNPs) (Table 2).

## 1.5. Polylactic-co-glycolic acid (PLGA)

PLGA nanoparticles can transport compounds across the round window membrane (RWM) into the perilymph, thereby increasing the local bioavailability of the encapsulated compound within the inner ear. After crossing the RWM and reaching the cochlea, these PLGA molecules are readily internalised by the cellular components of the organ of Corti (Youm et al., 2017). This ability enables PLGA nanoparticles to achieve gene silencing in a dose-dependent manner, even within the sensory epithelia of the cochlea (Du et al., 2013; Cai et al., 2014). Du et al. used poly(lactide-co-glycolide) PLGA nanoparticles to deliver siRNAs targeting the Hairy and Enhancer of Split 1 (Hes1) gene, a key player in the Notch signalling pathway. These nanoparticles have previously been used in the organs of Corti in P3 mice, with a transfection efficiency of approximately 45% (Du et al., 2013). In young adult guinea pigs, a micropump has been implanted at the basal turn of the cochlea 72 h after acoustic trauma (125 dB SPL noise for 3 h) and left in place for 24 h (Du et al., 2018); in the treated group, hearing is restored for up to 9 weeks, and Hes1 gene silencing via siRNA targeting results in partial restoration of outer hair cell (OHC) and inner hair cell (IHC) counts, as well as the presence of immature hair cells. These results suggest that the use of PLGA nanoparticles for siRNA delivery targeting the Hes1 gene may have potential as a therapeutic strategy for hearing restoration.

## 1.6. Polyhydroxyethyl aspartamide (PHEA)

Yoon et al. developed a graft copolymer by conjugating an oligoarginine peptide (Arg8) to a poly(amino acid) (poly(2-hydroxyethyl-l-aspartamide; PHEA)) (Yoon et al., 2015) and performed in vivo testing on mice at P30, where 20 µL of the PHEA-g-C18-Arg8 complex was placed on the round window via a gelatine sponge. After 24 h of evaluation, nanoparticles (with CMV-EGFP DNA fragment (1762 bp)) were observed in both inner hair cells (IHCs) and supporting cells, which were mainly concentrated in the modiolus and lateral wall tissues. GFP reporter gene expression was detected in the modiolus and lateral wall but not in the hair cells.

## 1.7. Polyethyleneimine (PEI) and dendrimers

A dendrimer-based nanocarrier has been developed and tested in vivo (Wu et al., 2013). The CMAP dendriplex (Na-carboxymethyl-beta-cyclodextrin-modified activated polyamidoamine dendrimers), which is associated with a plasmid containing Atoh1 (a transcription factor involved in the differentiation of sensory hair cells) and eGFP, was coupled with an absorbable gelatine sponge and applied to the round window of adult rats. At 7 days after application, the efficiency of transfection, as indicated by the expression of GFP, reached 48 ± 7% for the inner hair cell group and 82 ± 10% for the outer hair cell group. However, expression was not assessed beyond the first week. The researchers highlighted the deterioration of the auditory brainstem response (ABR) when polyethyleneimine (PEI) was used, whereas there was no significant difference in ABR deterioration between the CMAP dendriplex group and the control group.

Linear polyethyleneimine (PEI) has also been tested in guinea pigs via cochleostomy with the placement of an osmotic pump (Tan et al.,

<caption>**Table 2**</caption>

Summary of polymeric nanoparticle transfection methods and results in inner ear cells (Ref.: reference, Effic: efficiency, ABR: auditory brainstem response, Tox.: toxicity, RWM: round window membrane, OHC: outer hair cell, IHC: inner hair cell).

<table><thead><tr><th>Ref.</th><th>Vector</th><th>Trans-gene</th><th>Animal</th><th>Transformation age</th><th>Delivery</th><th>Analysis delay (days)</th><th>Targeted cells</th><th>Effic.</th><th>ABR</th><th>Tox.</th></tr></thead><tbody><tr><td>(Tan et al., 2008)</td><td>Polyethylenimine (PEI)</td><td>eGFP</td><td>Guinea pig</td><td>adult</td><td>cochleostomy middle turn minipump</td><td>7</td><td>vestibular and tympanic fibrocytes, Reissner's membrane mesenchymal and epithelial cells, and spiral ligament fibrocytes</td><td>-</td><td>-</td><td>-</td></tr><tr><td>(Zhang et al., 2011)</td><td>Hyperbranched polylysine nanoparticles</td><td>eGFP</td><td>Rat</td><td>P1-P5; adult</td><td>in vitro and gelatin sponge - RWM</td><td>1</td><td>OHC, IHC, supporting cells, spiral ganglion, stria vascularis, spiral ligament</td><td>9%</td><td>-</td><td>No</td></tr><tr><td>(Du et al., 2013)</td><td>poly(lactic-co-glycolic acid) PLGA</td><td>Hes1<br>Hes5</td><td>Mouse</td><td>P3</td><td>in vitro</td><td>1,2,3</td><td>OC cells</td><td>45%</td><td>-</td><td>-</td></tr><tr><td rowspan="2">(Wu et al., 2013)</td><td>Polyethylenimine (PEI)</td><td>Atoh1-eGFP</td><td>Rat</td><td>adult</td><td>gelatin sponge - RWM</td><td>7</td><td>OHC and IHC</td><td>0%</td><td>Lower</td><td>-</td></tr><tr><td>CM-β-CD modified activated PAMAM</td><td>Atoh1-eGFP</td><td>Rat</td><td>adult</td><td>gelatin sponge - RWM</td><td>7</td><td>OHC<br>IHC</td><td>48%<br>82%</td><td>As control<br>As control</td><td>-<br>-</td></tr><tr><td>(Kim et al., 2015)</td><td>poly(2-hydroxyethyl aspartamide) (PHEA)</td><td>Nile red</td><td>C57/BL6 mice</td><td>1 month</td><td>gelatin sponge - RWM</td><td>1</td><td>supporting cells</td><td>-</td><td>Lower DPOAE</td><td>Yes</td></tr><tr><td>(Youm et al., 2017)</td><td>poly D,L-daltic-co-glycolic acid (PLGA)</td><td>MAPK1 siRNA</td><td>Mouse</td><td>P3</td><td>in vitro</td><td>2</td><td>OHC</td><td>-</td><td>-</td><td>No</td></tr><tr><td>(Du et al., 2018)</td><td>poly(lactic-co-glycolic acid) PLGA</td><td>siRNA</td><td>Guinea pig</td><td>young adult</td><td>cochleostomy basal turn osmotic pump</td><td>21-64</td><td>OHC, IHC</td><td>-</td><td>Better</td><td>No</td></tr><tr><td>(Yang et al., 2018)</td><td>cationique polyethyleneglycol</td><td>-</td><td>C57/BL6</td><td>1 month</td><td>Middle ear</td><td>1-7</td><td>OHC, IHC</td><td>-</td><td>Normal</td><td>No</td></tr><tr><td>(Miwa et al., 2021)</td><td>vitamin E-scaffold SS-cleavable proton-activated lipid-like material (ssPalm)</td><td>eGFP<br>BDNF</td><td>Guinea pig</td><td>adult</td><td>gelatin sponge - RWM</td><td>28</td><td>Spiral ganglion neuron</td><td>Yes</td><td>Normal</td><td>Yes</td></tr></tbody></table>
modify the genetic material of cells to achieve a variety of therapeutic goals, including correcting disease-causing genetic mutations, producing specific proteins to treat specific diseases, and regulating gene expression.

The understanding of the ability of pulsed electric fields to transfer DNA across the plasma membrane has recently advanced. Studies have shown that the term 'electroporation' is not accurate, as plasmid DNA is too bulky to enter cells through the temporary pores created in the plasma membrane. Plasmid DNA, unlike small oligonucleotide ssDNA and RNA molecules, is internalized by cells primarily through active processes. The importance of the sequence of electrical pulses that achieves this 'electroporation' is to generate an electric field of sufficient intensity to physically bind negatively charged plasmid DNA to the cell surface facing the cathode, from which the pulses are emitted. This allows the DNA molecules to attach to the plasma membrane and subsequently be internalized by endocytosis and pinocytosis mechanisms (Escoffre et al., 2011; Rosazza et al., 2016). The majority of exogenous DNA is degraded within lysosomes and by endonuclease activity. However, some plasmids are transported to nuclear pores, exit transport vesicles, and become extrachromosomal (episomal) DNA (Pinyon et al., 2019).

This method of gene transfer should be called 'DNA electrotransfer' rather than 'electroporation. This method occurs due to the capacity of DNA to withstand the process without degradation and its ability to maintain expression for many months after translation has been detected within a few hours. The key parameters for efficient expression of recombinant proteins via DNA electrotransfer are related primarily to the intrinsic biophysics of the gene delivery process. These parameters include the initial concentration of plasmid DNA and the intensity and duration of the electric field. Plasmids are typically delivered to cells at uniform concentrations of microgram per microlitre. To generate voltage gradients, the parameters of the electrical pulses used for gene delivery are typically on the order of several hundred volts for tens of milliseconds (Rosazza et al., 2016). However, the need for high voltages in conventional electroporation may cause tissue damage due to irreversible dielectric breakdown and increased temperature (Lacković et al., 2009).

As shown in Table 3, most studies have been conducted in mice, with cell transfection rates ranging from 26 % to 98 % (Woods et al., 2004; Pinyon et al., 2014; Takeda et al., 2019). All cell types can be transfected, including hair cells, supporting cells, vascular stripe cells, and spiral ganglion cells (Gubbels et al., 2008; Brigande et al., 2009; Miwa et al., 2013). Gubbels reported that transfection is more effective at the base of the cochlea than in the middle. In the three studies that examined auditory thresholds, the technique did not induce hearing loss, and no toxicity was reported; however, the immune and inflammatory responses were not evaluated.

One of the advantages of this technology is that it can be applied to mouse embryos, allowing the effects of correcting an abnormality to be assessed at an early age, which is particularly important for studying the role of growth factors. Animal models for in utero gene therapy are currently being developed. Several studies have shown that when plasmids are delivered to the otocysts of embryonic mice at 11 days (E11), proteins are expressed in a significant proportion of cells and persist for up to 30 days after delivery (Gubbels et al., 2008). In a proof-of-concept study, in utero electroporation has been used to rescue auditory deficits by reintroducing Gjb6 and downregulating Gjb6 expression via short-strand RNAs (Miwa et al., 2013). Correcting genetic mutations in human embryos in utero may be considered in the future, providing that several technical barriers can be overcome, but this type of intervention to solve functional problems remains highly controversial, as the risk to the foetus cannot be completely eliminated (Müller and Barr-Gillespie, 2015).

## 1.11. Cochlear implant-mediated gene delivery

The use of near-field electroporation through the placement of a cochlear implant represents a hybrid technique with a unique principle (Pinyon et al., 2019), potentially combining current functional treatment with neurotrophic or even aetiological treatment (Pinyon et al., 2014). In a previous study, electrodes inserted into the cochlea of guinea pigs delivered electrical pulses to the surrounding tissue, which caused a temporary opening of the cell membrane, allowing genetic material to enter the mesenchymal cells that line the scala tympani. Once in the cell nucleus, the transferred BDNF gene produced brain-derived neurotrophic factor, which facilitated neurite regeneration within the cochlea, thereby correcting hypoacusis in a deaf guinea pig model (animals with damaged hair cells). As cochlear implants are widely used in patients with severe to profound deafness, the adjunctive use of electroporation may enable the use of gene therapy as a complementary means of treating hypoacusis. This could help improve patient understanding, particularly in noisy environments, which is a significant limitation of current therapeutic approaches.

## 1.12. Inorganic nanoparticles

### 1.12.1. Gene gun

The 'gene gun' is a gene transfer method in which small particles coated with genes are accelerated at high speeds towards target cells (Uchida et al., 2009). Gold and tungsten particles are commonly used as gene carriers because of their high density and small diameter, which allow them to effectively penetrate cells. Particle bombardment relies on the use of compressed gas, such as helium, to propel these particles towards the target cells (Belyantseva, 2009). An advantage of this technique is that it is not dependent on the specific characteristics of the target cells, including surface receptors or their electrical potential. The 'gene gun' also allows precise control of the gene transfer area. However, this technique can potentially alter cells due to the impact of accelerated particles. Finally, the use of compressed gas to accelerate particles requires specific equipment.

Belyantseva et al. used a gold-particle 'gene gun' to transfect hair cells from shaker 2 (Myo15a(sh2)) deaf mice with eGFP-Myo15a in vitro, restoring the normal pattern of hair bundles by recruiting endogenous Whirlin to the tips of stereocilia (Belyantseva et al., 2005).

The method has subsequently been refined to allow the transfection of hair cells from the basal side, thereby reducing the morphological changes induced by the technology (Zhao et al., 2012). However, to the best of our knowledge, no publication has yet tested this procedure in vivo.

### 1.12.2. Other inorganic nanoparticles

In addition to gold particles, other inorganic particles, such as silica, have been used for gene therapy in the inner ear (Wang et al., 2014; Schmidt et al., 2018; Guigou et al., 2021; Xu et al., 2021). In particular, mesoporous silica nanoparticles have been successfully used in vitro to deliver siRNA to the organ of the Corti cell line HEI-OC1 (Chen et al., 2022). The transfer efficiency was superior to that of Lipofectamine 3000, with no detectable toxicity, and the nanoparticles were absorbed onto the surface of electrode arrays of cochlear implants. Chen et al. demonstrated that siRNA can be released from inserted electrode arrays and diffuse into the inner ear after cochlear implantation. To our knowledge, no in vivo study of this type of inorganic nanoparticle has been reported at present.

Optimizing targeting precision is crucial not only for improving the efficacy of nanoparticles (NPs) but also for minimising potential side effects caused by techniques that lack cellular targeting. In the inner ear, hair cells and supporting cells play specific roles in sound transduction. Nonspecific targeting can disrupt these functions, reducing the effectiveness of therapy and potentially worsening hearing loss. For example, cadherin 23 is expressed exclusively in inner hair cells (IHCs) and outer
hair cells (OHCs) but not in supporting cells (Nishio et al., 2015). Thus, the expression of cadherin 23 in other cell types may interfere with sound transduction. Targeted delivery reduces the off-target effects of nanoparticles and limits the amount of genetic material and nanoparticles needed, thereby improving therapeutic safety and efficacy (Foster et al., 2024).

### 1.12.3. Specificity of nanoparticle targeting

Several strategies have been developed to improve the specificity of nanoparticle targeting, including modification of surface of nanoparticles with specific ligands (such as antibodies or peptides) that recognize and bind to specific receptors on target cells (Foster et al., 2024). For example, PEGylated PLA nanoparticles coated with the A666 peptide show greater targeting efficiency than untargeted nanoparticles, as evidenced by increased localization in outer hair cells and modiolus (Wang et al., 2018). Kayyali et al. reported that PEGylated liposomes coated with a prestin-targeting peptide present improved cochlear targeting and that targeted liposomes reach the apical turn, with both TdTdTdT and carboxyfluorescein being expressed in outer hair cells and other tissues, unlike untargeted liposomes (Kayyali et al., 2018). The drawback of this type of peptide-based targeting is the risk of an immune response upon readministration. With respect to the use of antibodies, Glueckert et al. revealed that nanoparticles conjugated with agonistic antibodies effectively activate TrkB receptors and enhance binding to inner ear cells, whereas lipid-based nanocapsules loaded with rolipram successfully prevent cisplatin-induced apoptosis, demonstrating their potential as drug delivery vectors (Glueckert et al., 2015; Foster et al., 2024).

To achieve more specific nanoparticle targeting, another alternative is the use of superparamagnetic iron oxide nanoparticles (SPIONs) with diameters greater than 50 nm (60–250 nm) (Xu et al., 2021). The experimental variables define the properties of the SPIONs, such as size, saturation magnetisation, surface charge, biocompatibility, and stability (Martin et al., 2024). Regarding systemic treatment, liposomes can protect SPIONs from degradation by enzymes in the blood and increase bioavailability in the circulation. A protein (e.g., a receptor-specific ligand) can be coupled to the liposomal bilayers to achieve specific cellular recognition (Safinya and Ewert, 2012; Guigou et al., 2021). In oncology, anti-CD20 SPIONs coated with liposomes, PEG, or Tween 80 have been used and tested in vitro against primary central nervous system lymphomas (Saesoo et al., 2018). PLGA coupled with iron oxide nanoparticles deposited on the RWM can be moved under the control of an external magnetic field for targeted entry into the perilymph (Ge et al., 2007). Although SPIONs themselves cannot carry genetic material, their encapsulation in PLGA allows the formation of a composite nanoparticle, thereby improving targeting precision (Liu and Yang, 2022).

Another approach to improve the efficacy of nanoparticles is to use hybrid vectors by coupling them with viral particles. Modified viral particles may aid in targeting specific cell types in the inner ear while coupling with nanoparticles to stabilize and protect the genetic material and limit immune side effects. This hypothesis has been successfully tested using herpes simplex virus glycoprotein H coupled to cationic lipids (Tu and Kim, 2008) or influenza virus (Wagner et al., 1992). For adeno-associated viruses, Mizuno et al. reported that the use of AAVs in combination with multilayer liposomes results in an approximately sixfold improvement in efficacy for oncological applications, particularly in the treatment of glioblastoma (Mizuno and Yoshida, 1998). Other options involving the encapsulation of whole viral particles have also been successfully tested (Fu and Zhang, 2001; Fein et al., 2009; Fiandaca et al., 2009). Although these solutions are promising, none have been tested in the inner ear.

Finally, optimizing the size and charge of nanoparticles plays an important role in targeting. The size and charge of nanoparticles influence their ability to penetrate cell membranes and be internalized by specific cells (Leong et al., 2018). For example, nanometre-sized

nanoparticles (< 100 nm) are more effective at penetrating hair cells (Glueckert et al., 2015).

### 1.12.4. Prospects and challenges

Currently, the nonviral vectors closest to clinical application are cationic lipids. In combination with a genome editing system, these methods restore hearing in mice with autosomal dominant deafness for at least 8 weeks (Gao et al., 2018; Tao et al., 2023). There are two main hypotheses regarding the loss of hearing after 4 months as follows: 1) although neonatal, the injection is too late; and 2) owing to the low number of edited cells (approximately 20%), the overall degeneration prevents these cells from maintaining sufficient auditory thresholds. To obtain these results, the injections were performed in mice at an early age, which, in terms of development, would correspond to an in utero injection in humans. In addition, the injections were made through 3 separate cochleostomies, the morbidity of which is likely not negligible in humans. The specificity of cationic lipids has been found to be low, which can be considered both an advantage and a disadvantage in certain cases to limit the risk of off-target effects associated with genome-modifying agents. Several targeting methods have been tested to improve the selection of transfected cells and to increase nuclear import (Tu and Kim, 2008; Le Guen et al., 2021). In addition, biocompatibility and the potential presence of an immune response need to be assessed before any clinical application.

Nanoparticles, a more recent development, have been tested in different age groups with mixed results. The tolerance profiles are generally good, with no toxicity reported in several studies (Zhang et al., 2011; Youm et al., 2017; Du et al., 2018; Yang et al., 2018). To our knowledge, only one study has reported auditory benefits. In a guinea pig model exposed to acoustic trauma, an approximate gain of 10 dB following the use of nanoparticles (PLGA) coupled to a transgene (siRNA targeting Hes1) was achieved (Du et al., 2018), revealing a sustained gain of hearing up to 9 weeks after surgery. Although moderate, these results are encouraging, particularly for the treatment of hearing loss due to acoustic trauma. However, the need for a cochleostomy may remain an obstacle to its clinical use for this purpose.

Electroporation has good results in terms of effectiveness on the organ of Corti, especially in the short term; however, Data beyond 7 days are limited (Gubbels et al., 2008; Miwa et al., 2013; Pinyon et al., 2014). A major advantage of this technique is that it can be used at an embryonic stage, allowing intervention before cochlear maturation. In a model of connexin 30 deafness, Miwa et al. restored hearing thresholds (approximately 50 dB) by transferring the wild-type gene for this connexin (Miwa et al., 2013). Although this success is theoretically not transferable to humans, it confirms the justified interest in gene therapy, especially for connexin-related deafness. Furthermore, Pinyon et al. reported an improvement in implant performance after the transfer of a neurotrophic factor by electroporation (Pinyon et al., 2014), but no transgene expression was detected after 10 weeks. Although performance is improved, the auditory gain associated with electroporation of neurotrophic factors has not been measured with ABR. Furthermore, the risks associated with the use of electrical pulses should be further investigated.

A new system of interest in gene therapy is plasmid vector systems with scaffold/matrix attachment regions (S/MARs). S/MARs are AT-rich genomic sequences that anchor chromatin to nuclear matrix proteins, playing a role in gene regulation. When incorporated into plasmid DNA vectors, S/MARs enhance episomal maintenance and mitotic stability, enabling sustained gene expression both in vitro and in vivo. By testing vectors in HEK293 cells, patient-derived fibroblasts, and zebrafish models, Toms et al. demonstrated sustained gene expression, restoration of usherin, and functional rescue of the usher 2 complex in photoreceptors (Toms et al., 2023). This is the first reported vector capable of expressing full-length usherin, revealing prospects for its use in the cochlea.

In conclusion, LNs offer no size limitations and have low
immunogenicity and compatibility with genome editing technologies. However, the efficacy of LNPs is variable, and there are concerns about long-term effects and potential OHC toxicity. Polymeric nanoparticles offer good efficiency and can cross the round window membrane with encapsulated compounds, but they may cause side effects or hearing loss. Electroporation allows targeted delivery without cell selection and can be combined with a cochlear implant, but it carries a risk of tissue damage. Inorganic nanoparticles offer precise control of the gene delivery site but can cause tissue damage, and in vivo data on inorganic nanoparticles are lacking.

Nonviral vectors hold great promise for treating the genetic causes of hearing loss. While progress has been made in animal models, overcoming challenges related to long-term efficacy, potential toxicity, and clinical translation is essential to make these therapies available to deaf patients.

Acknowledgments: Thanks to Gemma Davies and Jacob Kachura for their precise proofreading and English corrections. This work was supported by grants from "Association de transfusion sanguine et de bio-génétique Gaetan Saleün" (Brest, France), "Région Bretagne" and Brest Métropole.

## CRediT authorship contribution statement

**Jean-Christophe Leclère:** Writing – review & editing, Writing – original draft, Visualization, Resources, Methodology, Investigation, Conceptualization.
**Remi Marianowski:** Writing – review & editing, Writing – original draft, Validation, Project administration, Methodology, Formal analysis, Conceptualization.
**Tristan Montier:** Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Funding acquisition, Conceptualization.

## Data availability

Data will be made available on request.

## References

Afonso, D., Le Gall, T., Couthon-Gourvès, H., Grélard, A., Prakash, S., Berchel, M., et al., 2016. Triggering bilayer to inverted-hexagonal nanostructure formation by thiol-ene click chemistry on cationic lipids: consequences on gene transfection. Soft. Matter. 12, 4516–4520. https://doi.org/10.1039/c6sm00609d.

Akil, O., Dyka, F., Calvet, C., Emptoz, A., Lahlou, G., Nouaille, S., et al., 2019. Dual AAV-mediated gene therapy restores hearing in a DFN89 mouse model. Proc. Natl. Acad. Sci. U.S.A 116, 4496–4501. https://doi.org/10.1073/pnas.1817537116.

Al-Moyed, H., Cepeda, A.P., Jung, S., Moser, T., Kügler, S., Reisinger, E., 2019. A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf ototeflin knock-out mice. EMBO Mol. Med. 11, e9396. https://doi.org/10.15252/emmb.201809396.

Belmadi, N., Midoux, P., Loyer, P., Passirani, C., Pichon, C., Le Gall, T., et al., 2015. Synthetic vectors for gene delivery: an overview of their evolution depending on routes of administration. Biotechnol. J. 10, 1370–1389. https://doi.org/10.1002/biot.201400841.

Belyantseva, I.A., 2009. Helios Gene Gun-mediated transfection of the inner ear sensory epithelium. Methods Mol. Biol. 493, 103–123. https://doi.org/10.1007/978-1-59745-523-7-7.

Belyantseva, I.A., Boger, E.T., Friedman, T.B., 2003. Myosin XVa localizes to the tips of inner ear sensory cell stereocilia and is essential for staircase formation of the hair bundle. Proc. Natl. Acad. Sci. U.S.A 100, 13958–13963. https://doi.org/10.1073/pnas.2334417100.

Belyantseva, I.A., Boger, E.T., Naz, S., Frolenkov, G.I., Sellers, J.R., Ahmed, Z.M., et al., 2005. Myosin-XVa is required for tip localization of whirli and differential elongation of hair-cell stereocilia. Nat. Cell Biol. 7, 148–156. https://doi.org/10.1038/ncb1219.

Billiet, L., Gomez, J.-P., Berchel, M., Jaffrès, P.-A., Le Gall, T., Montier, T., et al., 2012. Gene transfer by chemical vectors and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line. Biomaterials 33, 2980–2990. https://doi.org/10.1016/j.biomaterials.2011.12.027.

Brigande, J.V., Gubbels, S.P., Woessner, D.W., Jungwirth, J.J., Bresee, C.S., 2009. Electroporation-mediated gene transfer to the developing mouse inner ear. Methods Mol. Biol. 493, 125–139. https://doi.org/10.1007/978-1-59745-523-7-8.

Cai, H., Wen, X., Wen, L., Tirelli, N., Zhang, X., Zhang, Y., et al., 2014. Enhanced local bioavailability of single or compound drugs delivery to the inner ear through application of PLGA nanoparticles via round window administration. Int. J. Nanomed. 9, 5591–5601. https://doi.org/10.2147/IJN.S72555.

Casey, G., Askew, C., Brimble, M.A., Samulski, R.J., Davidoff, A.M., Li, C., et al., 2020. Self-complementarity in adeno-associated virus enhances transduction and gene expression in mouse cochlear tissues. PLoS One 15, e0242599. https://doi.org/10.1371/journal.pone.0242599.

Chen, A., Chen, Y., Liu, S., Ma, D., Tang, J., Zhang, H., 2022. Mesoporous silica nanoparticle-modified electrode arrays of cochlear implants for delivery of siRNA-TGFβ1 into the inner ear. Colloids. Surf. B Biointerfaces. 218, 112753. https://doi.org/10.1016/j.col surfb.2022.112753.

Delmaghani, S., El-AmrEOUI, A., 2020. Inner ear gene therapies take off: current promises and future challenges. J. Clin. Med. 9, 2309. https://doi.org/10.3390/jcm9072309.

Du, X., Cai, Q., West, M.B., Youm, I., Huang, X., Li, W., et al., 2018. Regeneration of cochlear hair cells and hearing recovery through Hes1 modulation with siRNA nanoparticles in adult guinea pigs. Mol. Ther. 26, 1313–1326. https://doi.org/10.1016/j.ymthe.2018.03.004.

Du, X., Li, W., Gao, X., West, M.B., Saltzman, W.M., Cheng, C.J., et al., 2013. Regeneration of mammalian cochlear and vestibular hair cells through Hes1/Hes5 modulation with siRNA. Hear. Res. 304, 91–110. https://doi.org/10.1016/j.heares.2013.06.011.

Escoffre, J.-M., Portet, T., Favard, C., Teissié, J., Dean, D.S., Rols, M.-P., 2011. Electromigrated formation of DNA complexes with cell membranes and its consequences for gene delivery. Biochim. Biophys. Acta 1808, 1538–1543. https://doi.org/10.1016/j.bbamem.2010.10.009.

Fein, D.E., Limberis, M.P., Maloney, S.F., Heath, J.M., Wilson, J.M., Diamond, S.L., 2009. Cationic lipid formulations alter the in vivo tropism of AAV2/9 vector in lung. Mol. Ther. 17, 2078–2087. https://doi.org/10.1038/mt.2009.173.

Fiandaca, M.S., Varenika, V., Eberling, J., McKnight, T., Bringas, J., Pivirotto, P., et al., 2009. Real-time MR imaging of adeno-associated viral vector delivery to the primate brain. Neuroimage 2 (47 Suppl), T27–T35. https://doi.org/10.1016/j.neuroimage.2008.11.012.

Foster, T., Lim, P., Jones, M., Wagle, S.R., Kovacevic, B., Ionescu, C.M., et al., 2024. Polymer-based nanoparticles for inner ear targeted trans differentiation gene therapy. ChemMedChem., e202400038. https://doi.org/10.1002/cmdc.202400038.

Fu, X., Zhang, X., 2001. Delivery of herpes simplex virus vectors through liposome formulation. Mol. Ther. 4, 447–453. https://doi.org/10.1006/mthe.2001.0474.

Gao, X., Tao, Y., Lamas, V., Huang, M., Yeh, W.-H., Pan, B., et al., 2018. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature 553, 217–221. https://doi.org/10.1038/nature25164.

Ge, X., Jackson, R.L., Liu, J., Harper, E.A., Hoffer, M.E., Wassel, R.A., et al., 2007. Distribution of PLGA nanoparticles in chinchilla cochleae. Otolaryngol. Head. Neck. Surg. 137, 619–623. https://doi.org/10.1016/j.otohns.2007.04.013.

Gélécot, G.S.G., Holt, J.R., 2014. Sound strategies for hearing restoration. Science (1979) 344, 1241062. https://doi.org/10.1126/science.1241062.

Ghosh, A., Yue, Y., Duan, D., 2011. Efficient transgene reconstitution with hybrid dual AAV vectors carrying the minimized bridging sequences. Hum. Gene Ther. 22, 77–83. https://doi.org/10.1089/hum.2010.122.

Glueckert, R., Pritz, C.O., Roy, S., Dudas, J., Schrott-Fischer, A., 2015. Nanoparticle mediated drug delivery of rolipram to tyrosine kinase B positive cells in the inner ear with targeting peptides and agonistic antibodies. Front. Aging Neurosci. 7, 71. https://doi.org/10.3389/fnagi.2015.00071.

Gubbels, S.P., Woessner, D.W., Mitchell, J.C., Ricci, A.J., Brigande, J.V., 2008. Functional auditory hair cells produced in the mammalian cochlea in utero gene transfer. Nature 455, 537–541. https://doi.org/10.1038/nature07265.

Guigou, C., Lalande, A., Millot, N., Belharet, K., Bozorg Grayeli, A., 2021. Use of super paramagnetic iron oxide nanoparticles as drug carriers in brain and ear: state of the art and challenges. Brain Sci. 11, 358. https://doi.org/10.3390/brainsci11030358.

Isgir, K., McDougald, D.S., Zhu, J., Wang, H.J., Bennett, J., Chien, W.W., 2019. AAV2.7m8 is a powerful viral vector for inner ear gene therapy. Nat. Commun. 10, 427. https://doi.org/10.1038/s41467-018-08243-1.

Ishibashi, Y., Sung, C.Y.W., Grati, M., Chien, W., 2023. Immune responses in the mammalian inner ear and their implications for AAV-mediated inner ear gene therapy. Hear. Res. 432, 108735. https://doi.org/10.1016/j.heares.2023.108735.

Jero, J., Mhatre, A.N., Tseng, C.J., Stern, R.E., Coling, D.E., Goldstein, J.A., et al., 2001a. Cochlear gene delivery through an intact round window membrane in mouse. Hum. Gene Ther. 12, 539–548. https://doi.org/10.1089/104303401300042465.

Jero, J., Tseng, C.J., Mhatre, A.N., Lalwani, A.K., 2001b. A surgical approach appropriate for targeted cochlear gene therapy in the mouse. Hear. Res. 151, 106–114. https://doi.org/10.1016/S0378-5955(00)000216-1.

Kayyali, M.N., Wooltorton, J.R.A., Ramsey, A.J., Lin, M., Chao, T.N., Tsourkas, A., et al., 2018. A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss. J. Control Release 29, 243–250. https://doi.org/10.1016/j.jconrel.2018.04.028.

Kim, D.-K., Park, S.-N., Park, K.-H., Park, C.W., Yang, K.-J., Kim, J.-D., et al., 2015. Development of a drug delivery system for the inner ear using poly(amino acid)-based nanoparticles. Drug Deliv. 22, 367–374. https://doi.org/10.3109/10717544.2013.879354.

Klein, N.P., Lewis, N., Goddard, K., Fireman, B., Zerbo, O., Hanson, K.E., et al., 2021. Surveillance for adverse events after COVID-19 mRNA vaccination. JAMA 326, 1390–1399. https://doi.org/10.1001/jama.2021.15072.

Lacković, I., Magjarević, R., Miklavčić, D., 2009. Three-dimensional finite-element analysis of joule heating in electrochemotherapy and in vivo gene Electrotransfer. IEEE Trans. Dielectric. Electric. Insulat. 16, 1338–1347. https://doi.org/10.1109/TDEI.2009.5293947.

Le Corre, S.S., Berchel, M., Belmadi, N., Denis, C., Haelters, J.-P., Le Gall, T., et al., 2014. Cationic lipophosphoramidates with two different lipid chains: synthesis and evaluation as gene carriers. Org. Biomol. Chem. 12, 1463–1474. https://doi.org/10.1039/c3ob42270d.
Le Guen, Y.T., Pichon, C., Guégan, P., Pluchon, K., Haute, T., Quemener, S., et al., 2021. DNA nuclear targeting sequences for enhanced non-viral gene transfer: an in vitro and in vivo study. Mol. Ther. Nucleic. Acids. 24, 477–486. https://doi.org/10.1016/j.omtn.2021.03.012.

Lee, S., Dondzillo, A., Gubbels, S.P., Raphael, Y., 2020. Practical aspects of inner ear gene delivery for research and clinical applications. Hear. Res. 394, 107934. https://doi.org/10.1016/j.heares.2020.107934.

Leong, J., Teo, J.Y., Aakalu, V.K., Yang, Y.Y., Kong, H., 2018. Engineering polymersomes for diagnostics and therapy. Adv. Healthc. Mater. 7, e1701276. https://doi.org/10.1002/adhm.201701276.

Li, X., Burn, E., Duarte-Salles, T., Yin, C., Reich, C., Delmestri, A., et al., 2022. Comparative risk of thrombosis with thrombocytopenia syndrome or thromboembolic events associated with different covid-19 vaccines: international network cohort study from five European countries and the US. BMJ 379, e071594. https://doi.org/10.1136/bmj.2022-071594.

Lindberg, M.F., Le Gall, T., Carmoy, N., Berchel, M., Hyde, S.C., Gill, D.R., et al., 2015. Efficient in vivo transfection and safety profile of a CpG-free and codon optimized luciferase plasmid using a cationic lipophosphoramidate in a multiple intravenous administration procedure. Biomaterials 59, 1–11. https://doi.org/10.1016/j.biomaterials.2015.04.024.

Liu, S.S., Yang, R., 2022. Inner ear drug delivery for sensorineural hearing loss: current challenges and opportunities. Front. Neurosci. 16, 867453. https://doi.org/10.3389/fnins.2022.867453.

Lv, J., Wang, H., Cheng, X., Chen, Y., Wang, D., Zhang, L., et al. (2024). AAV1-hTOF gene therapy for autosomal recessive deafness 9: a single-arm trial. *The Lancet* 0. doi: 10.1016/S0140-6736(23)02874-X.

Maeda, Y., Fukushima, K., Kawasaki, A., Nishizaki, K., Smith, R.J.H., 2007. Cochlear expression of a dominant-negative GJB2R75W construct delivered through the round window membrane in mice. Neurosci. Res. 58, 250–254. https://doi.org/10.1016/j.neures.2007.03.006.

Martin, M.J., Montiel Schneider, M.G., Paolillo, G., Sanchez, F.H., Lo Fiego, M., Spitzmaul, G., et al., 2024. Development of magnetic nanosystems with potential for the treatment of inner ear pathologies. ChemMedChem, e204200321. https://doi.org/10.1002/cmdc.204200321.

Mehier-Humbert, S., Guy, R.H., 2005. Physical methods for gene transfer: improving the kinetics of gene delivery into cells. Adv. Drug Deliv. Rev. 57, 733–753. https://doi.org/10.1016/j.addr.2004.12.007.

Miwa, T., Minoda, R., Ise, M., Yamada, T., Yumoto, E, 2013. Mouse otocyst transuterine gene transfer restores hearing in mice with connexin 30 deletion-associated hearing loss. Mol. Ther. 21, 1142–1150. https://doi.org/10.1038/mt.2013.62.

Miwa, T., Saito, H., Akita, H., 2021. Lipid nanoparticles-encapsulated brain-derived neurotrophic factor mRNA delivered through the round window niche in the cochlea of guinea pigs. Exp. Brain Res. 239, 425–433. https://doi.org/10.1007/s00221-020-05970-0.

Mizuno, M., Yoshida, J, 1998. Improvement of transduction efficiency of recombinant
adeno-associated virus vector by entrapment in multilamellar liposomes. Jpn. J.
Cancer Res. 89, 352–354. https://doi.org/10.1111/j.1349-7006.1998.tb00570.x.

Mukherjee, D., Jajoo, S., Kaur, T., Sheehan, K.E., Ramkumar, V., Rybak, L.P., 2010. Transtympanic administration of short interfering (si)RNA for the NOX3 isoform of NADPH oxidase protects against cisplatin-induced hearing loss in the rat. Antioxid. Redox. Signal. 13, 589–598. https://doi.org/10.1089/ars.2010.3110.

Müller, U., Barr-Gillespie, P.G., 2015. New treatment options for hearing loss. Nat. Rev. Drug Discov. 14, 346–365. https://doi.org/10.1038/ndr4533.

Nishio, S.-Y., Hattori, M., Moteki, H., Tsukada, K., Miyagawa, M., Naito, T., et al., 2015. Gene expression profiles of the cochlea and vestibular endorgans: localization and function of genes致 deafness. Ann. Otol. Rhinol. Laryngol. 124 (1), 6S–48S. https://doi.org/10.1177/0003489415575549. https://doi.org/10.1016/j.bbagg.2009.05.013.

Philippidis, A., 2022. Novartis Confirms Deaths of Two Patients Treated with Gene Therapy Zolgensma. Hum. Gene Ther. 33, 842–844. https://doi.org/10.1089/hum.2022.29216.bfs.

Pinyon, J.L., Tadros, S.F., Froud, K.E., Wong, Y., A., C., Tompson, I.T., Crawford, E.N., et al., 2014. Close-field electroporation gene delivery using the cochlear implant electrode array enhances the bionic ear. Sci. Transl. Med. 6. https://doi.org/10.1126/scitranslmed.3008177, 233ra54.

Pinyon, J.L., von Jonquieres, G., Crawford, E.N., Duxbury, M., Al Abed, A., Lovell, N.H., et al., 2019. Neurotrophin gene augmentation by electrotransfer to improve cochlear implant hearing outcomes. Hear. Res. 380, 137–149. https://doi.org/10.1016/j.heares.2019.06.002.

Puligilla, C., Dabdoub, A., Brenowitz, S.D., Kelley, M.W., 2010. Sox2 induces neuronal formation in the developing mammalian cochlea. J. Neurosci. 30, 714–722. https://doi.org/10.1523/JNEUROSCI.3852-09.2010.

Purcell, P.L., Deep, N.L., Waltzman, S.B., Roland, J.T., Cushing, S.L., Papsin, B.C., et al., 2021. Cochlear implantation in infants: why and how. Trends. Hear. 25, 23312165211031751. https://doi.org/10.1177/23312165211031751.

Rosazza, C., Deschout, H., Buntz, A., Braeckmans, K., Rols, M.-P., Zumbusch, A., 2016. Endocytosis and endosomal trafficking of DNA after gene electrotransfer In Vitro. Mol. Ther. Nucleic. Acids. 5, e286. https://doi.org/10.1038/mtna.2015.59.

Saesoo, S., Sathornsumetee, S., Anekwiang, P., Treetidnipa, C., Thuwajit, P., Bunthot, S., et al., 2018. Characterization of liposome-containing SPIONs conjugated with anti-CD20 developed as a novel theranostic agent for central nervous system lymphoma. Colloids. Surf. B Biointeriors. 161, 497–507. https://doi.org/10.1016/j. colsurfb.2017.11.003.

Safinya, C.R., Ewert, K.K., 2012. Liposomes derived from molecular vases. Nature 489, 372–374. https://doi.org/10.1038/489372.

Scheper, V., Wolf, M., Scholl, M., Kadlecova, Z., Perrier, T., Klok, H.-A., et al., 2009. Potential novel drug carriers for inner ear treatment: hyperbranched polylysine and

lipid nanocapsules. Nanomedicine (Lond) 4, 623–635. https://doi.org/10.2217/nmi.09.41.

Schmidt, N., Schulze, J., Warwas, D.P., Ehlert, N., Lenarz, T., Warnecke, A., et al., 2018. Long-term delivery of brain-derived neurotrophic factor (BDNF) from nanoporous silica nanoparticles improves the survival of spiral ganglion neurons in vitro. PLoS. One 13, e0194778. https://doi.org/10.1371/journal.pone.0194778.

Smith, R.J.H., Bale, J.F., White, K.R., 2005. Sensorineural hearing loss in children. Lancet 365, 879–890. https://doi.org/10.1016/S0140-6736(05)71047-3.

Sobh, M., Lagali, P.S., Ghiasi, M., Montroy, J., Dollin, M., Hurley, B., et al., 2023. Safety and efficacy of adeno-associated viral gene therapy in patients with retinal degeneration: a systematic review and meta-analysis. Transl. Vis. Sci. Technol. 12, 24. https://doi.org/10.1167/tvst.2012.11.24.

Staecker, H., Li, D., O'Malley, B.W., Van De Water, T.R., 2001. Gene expression in the mammalian cochlea: a study of multiple vector systems. Acta Otolaryngol. 121, 157–163. https://doi.org/10.1080/000164801300043307.

Suhr, O.B., Coelho, T., Buades, J., Pouget, J., Conceicao, I., Berk, J., et al., 2015. Efficacy and safety of patisiran for familial amyloidotic polyneuropathy: a phase II multi-dose study. Orphanet. J. Rare Dis. 10, 109. https://doi.org/10.1186/s13023-015-0326-6.

Takeda, H., Miwa, T., Kim, M.Y., Choi, B.Y., Orita, Y., Minoda, R., 2019. Prenatal electroporation-mediated gene transfer restores Slc26a4 knock-out mouse hearing and vestibular function. Sci. Rep. 9, 17979. https://doi.org/10.1038/s41598-019-54262-3.

Tan, B.T.G., Foong, K.H., Lee, M.M.G., Ruan, R., 2008. Polyethylenimine-mediated cochlear gene transfer in guinea pigs. Arch. Otolaryngol. Head Neck Surg. 134, 884–891. https://doi.org/10.1001/archotol.134.8.884.

Tan, F., Chu, C., Qi, J., Li, W., You, D., Li, K., et al., 2019. AAV-ie enables safe and efficient gene transfer to inner ear cells. Nat. Commun. 10, 3733. https://doi.org/10.1038/s41467-019-11687-8.

Tao, Y., Lamas, V., Du, W., Zhu, W., Li, Y., Whittaker, M.N., et al., 2023. Treatment of monogenic and digenic dominant genetic hearing loss by CRISPR-Cas9 ribonucleoprotein delivery in vivo. Nat. Commun. 14, 4928. https://doi.org/10.1038/s41467-023-40476-7.

Toms, M., Toualbi, L., Almeida, P.V., Harbottle, R., Moosajee, M., 2023. Successful large gene augmentation of USH2A with non-viral episomal vectors. Mol. Ther. 31, 2755–2766. https://doi.org/10.1016/j.ymthe.2023.06.012.

Tu, Y., Kim, J., 2008. A fusogenic segment of glycoprotein H from herpes simplex virus enhances transfection efficiency of cationic liposomes. J. Gene Med. 10, 646–654. https://doi.org/10.1002/jgm.1184.

Uchida, M., Li, X.W., Mertens, P., Alpar, H.O., 2009. Transfection by particle bombardment: delivery of plasmid DNA into mammalian cells using gene gun. Biochim. Biophys. Acta 1790, 754–764. https://doi.org/10.1016/j.bbagg.2009.05.013.

Vreugde, S., Erven, A., Kros, C.J., Marcotti, W., Fuchs, H., Kurima, K., et al., 2002. Beethoven, a mouse model for dominant, progressive hearing loss DFNA36. Nat. Genet. 30, 257–258. https://doi.org/10.1038/ng848.

Wagner, E., Plank, C., Zatloukal, K., Cotten, M., Birnstiel, M.L., 1992. Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle. Proc. Natl. Acad. Sci. U.S.A 89, 7934–7938. https://doi.org/10.1073/pnas.89.17.7934.

Wang, C., Zhang, Y., Dong, Y., 2021. Lipid Nanoparticle-mRNA Formulations for Therapeutic Applications. Acc. Chem. Res. 54, 4283–4293. https://doi.org/10.1021/acs.accounts.1c00550.

Wang, X., Chen, Y., Tao, Y., Gao, Y., Yu, D., Wu, H., 2018. A666-conjugated nanoparticles target prestin of outer hair cells preventing cisplatin-induced hearing loss. Int. J. Nanomed. 13, 7517–7531. https://doi.org/10.1247/IJN.1S170130.

Wang, Y., Wise, A.K., Tan, J., Maina, J.W., Shepherd, R.K., Caruso, F., 2014. Mesoporous silica supraparticles for sustained inner-ear drug delivery. Small. 10, 4244–4248. https://doi.org/10.1002/smll.201401767.

Wareing, M., Mhatre, A.N., Pettis, R., Han, J.J., Haut, T., Pfister, M.H., et al., 1999. Cationic liposome mediated transgene expression in the guinea pig cochlea. Hear. Res. 128, 61–69. https://doi.org/10.1016/s0378-5955(98)00196-8.

Woods, C., Montcouquiol, M., Kelley, M.W., 2004. Math1 regulates development of the sensory epithelium in the mammalian cochlea. Nat. Neurosci. 7, 1310–1318. https://doi.org/10.1038/nn1349.

World report on hearing (2019). Available at: https://www.who.int/publications-detail-redirect/9789240020481 (Accessed May 3, 2024).

Wrobel, I., Collins, D, 1995. Fusion of cationic liposomes with mammalian cells occurs after endocytosis. Biochim. Biophys. Acta 1235, 296–304. https://doi.org/10.1016/0005-2736(95)80017-a.

Wu, N., Li, M., Chen, Z.-T., Zhang, X.-B., Liu, H.-Z., Li, Z., et al., 2013. In vivo delivery of Atoh1 gene to rat cochlea using a dendrimer-based nanocarrier. J. Biomed. Nanotechnol. 9, 1736–1745. https://doi.org/10.1166/jbn.2013.1684.

Xiong, W., Grillet, N., Elledge, H.M., Wagner, T.F.J., Zhao, B., Johnson, K.R., et al., 2012. TMHS is an integral component of the mechanotransduction machinery of cochlear hair cells. Cell 151, 1283–1295. https://doi.org/10.1016/j.cell.2012.10.041.

Xiong, W., Wagner, T., Yan, L., Grillet, N., Müller, U., 2014. Using injectoporation to deliver genes to mechanosensory hair cells. Nat. Protoc. 9, 2438–2449. https://doi.org/10.1038/nprot.2014.168.

Xu, X., Zheng, J., He, Y., Lin, K., Li, S., Zhang, N., et al., 2021. Nanocarriers for inner ear disease therapy. Front. Cell Neurosci. 15, 791573. https://doi.org/10.3389/fncel.2021.791573.

Yang, K.-J., Son, J., Jung, S.Y., Yi, G., Yoo, J., Kim, D.-K., et al., 2018. Optimized phospholipid-based nanoparticles for inner ear drug delivery and therapy. Biomaterials 171, 133–143. https://doi.org/10.1016/j.biomaterials.2018.04.038.
Yoon, J.Y., Yang, K.-J., Kim, D.E., Lee, K.-Y., Park, S.-N., Kim, D.-K., et al., 2015. Intratympanic delivery of oligoarginine-conjugated nanoparticles as a gene (or drug) carrier to the inner ear. *Biomaterials* 73, 243–253. https://doi.org/10.1016/j.biomaterials.2015.09.025.

Youm, I., West, M.B., Li, W., Du, X., Ewert, D.L., Kopke, R.D., 2017. siRNA-loaded biodegradable nanocarriers for therapeutic MAPK1 silencing against cisplatin-induced ototoxicity. *Int. J. Pharm.* 528, 611–623. https://doi.org/10.1016/j.ijpharm.2017.06.035.

Zhang, W., Zhang, Y., Löbler, M., Schmitz, K.-P., Ahmad, A., Pyykkö, I., et al., 2011. Nuclear entry of hyperbranched polylysine nanoparticles into cochlear cells. *Int. J. Nanomedicine* 6, 535–546. https://doi.org/10.2147/IJN.S16973.

Zhao, H., Avenarius, M.R., Gillespie, P.G., 2012. Improved biolistic transfection of hair cells. *PLoS One* 7, e46765. https://doi.org/10.1371/journal.pone.0046765.

Zheng, J.L., Gao, W.Q., 2000. Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. *Nat. Neurosci.* 3, 580–586. https://doi.org/10.1038/75753.

Zou, J., Saulnier, P., Perrier, T., Zhang, Y., Manninen, T., Toppila, E., et al., 2008. Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation. *J. Biomed. Mater. Res. B Appl. Biomater.* 87, 10–18. https://doi.org/10.1002/jbm.b.31058.

Zuris, J.A., Thompson, D.B., Shu, Y., Guilinger, J.P., Bessen, J.L., Hu, J.H., et al., 2015. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. *Nat. Biotechnol.* 33, 73–80. https://doi.org/10.1038/nbt.3081.