Deafness is commonly caused by the irreversible loss of mammalian cochlear hair cells (HCs) due to noise trauma, toxins, or infections. We previously demonstrated that small interfering RNAs (siRNAs) directed against the Notch pathway gene, hairy and enhancer of split 1 (Hes1), encapsulated within biocompatible poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) could regenerate HCs within ototoxin-ablated murine organotypic cultures. In the present study, we delivered this sustained-release formulation of Hes1 siRNA (siHes1) into the cochleae of noise-injured adult guinea pigs. Auditory functional recovery was measured by serial auditory brainstem responses over a nine-week follow-up period, and HC regeneration was evaluated by immunohistological evaluations and scanning electron microscopy. Significant HC restoration and hearing recovery were observed across a broad tonotopic range in ears treated with siHes1 NPs, beginning at three weeks and extending out to nine weeks post-treatment. Moreover, both ectopic and immature HCs were uniquely observed in noise-injured cochleae treated with siHes1 NPs, consistent with de novo HC production. Our results indicate that durable cochlear HCs were regenerated and promoted significant hearing recovery in adult guinea pigs through reversible modulation of Hes1 expression. Therefore, PLGA-NP-mediated delivery of siHes1 to the cochlea represents a promising pharmacologic approach to regenerate functional and sustainable mammalian HCs in vivo.

Previous studies have demonstrated that, in addition to maintaining lateral inhibition, Notch pathway gene expression is increased in mammalian cochleae and vestibular end organs (i.e., utricles and saccules) after acoustic trauma or ototoxic insults.In the adult guinea pig, ototoxic damage to the cochlea has been shown to result in elevated Hes1 and Hes5 expression in concert with increased expression of the Notch1 receptor and ligands, such as Jagged1, among surviving SCs.Acoustic traumas have also been shown to induce upregulation of Hes1 and Hes5 mRNA in the OC.Based on its central role in promoting and maintaining the SC phenotype, both elevated and homeostatic Notch-dependent Hes1 expression among surviving SCs may actively oppose an intrinsic HC regenerative program.Consistent with these reports, results from our previous in vitro studies and other labs have indicated that targeted and reversible silencing of Hes1 expression is sufficient to promote pro-transdifferentiative signaling in the postnatal murine cochlea.In the present study, we delivered biocompatible poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) containing Hes1 small interfering RNA (siRNA) payloads into the cochleae of adult guinea pigs to evaluate whether this refined pharmacological approach could also induce cochlear HC regeneration and hearing recovery in vivo in adult animals that had been exposed to a deafening noise trauma.

In the mammalian cochlea, HCs and supporting cells (SCs), which provide trophic, structural, and functional support to HCs, are arranged in an alternating mosaic-like pattern, in which adjacent HCs do not directly contact one another. This spatial specificity is established during embryonic development and is maintained postnatally through lateral inhibition via active Notch signaling. Notch is a cell surface receptor protein that, when activated, upregulates downstream effector molecules, such as hairy and enhancer of split-1 and 5 (Hes1 and Hes5), which in turn inhibit the expression of proneural basic helix-loop-helix transcription factors, such as atonal homolog 1 (Atoh1).HCs and SCs arise from a common prosensory epithelial cell lineage,and Atoh1 expression is critical for initiating the transcriptional cascade necessary for HC differentiation.Thus, lateral inhibition prevents SCs from transdifferentiating into HCs through active Notch signaling that is maintained via direct contact with Notch ligands expressed on the surfaces of adjacent HCs or neighboring SCs.Indeed, in nonmammalian vertebrates, SCs spontaneously regenerate into new HCs through both mitotic and non-mitotic (transdifferentiation) mechanisms.Studies have shown that this process can be opposed or reversed by Notch pathway activation.Conversely, other in vivo studies indicate that, in mammals, either decreasing Notch signaling within the organ of Corti (OC) or increasing Atoh1 expression is sufficient to generate new HCs through direct transdifferentiation of pre-existing SCs.

Regenerated hair cells can originate from supporting cell progeny: evidence from phototoxicity and laser ablation experiments in the lateral line system.

Deafness and loss of balance are commonly caused by a loss of sensory hair cells (HCs) due to toxins, infections, noise and/or blast trauma, aging, and other factors.Once lost, cochlear HCs in mammals do not spontaneously regenerate, which can result in permanent hearing impairments, as HC attrition accumulates over time or is acutely manifested following a severe acoustic or ototoxic trauma.

SC counts were also performed at defined tonotopic regions within noise-injured OCs from both scRNA-NP- and siHes1-NP-treated animals and compared to SC counts performed among age-matched normal controls. In undamaged OCs, SC densities in the OHC region were relatively uniform among the tonotopic sampling regions, ranging from an average of 357.5/mm at 1 kHz to 378.75/mm at 32 kHz. Overall, SC numbers were decreased across these sampling regions in the siHes1 NP treatment group compared to normal controls. However, significant differences in total SC numbers were observed only across the 32-kHz sampling region (p < 0.001; Figure 8 B). In contrast, the SC numbers were significantly reduced in the scRNA group in all three frequency regions compared to either normal controls or the siHes1 NP treatment group (p < 0.01 or 0.001; Figure 8 B). In these cochleae, a pronounced basal-to-apical gradient of SC loss was measured, such that the 32-kHz frequency region possessed approximately 49.4% of the number of SCs as the 1-kHz region (p < 0.01). These results seem to indicate that, in the absence of a viable OHC population, the OC continues to degenerate, eschewing SCs as it forms flat epithelia. Taken together, these results imply that siHes1 NP treatment restores HC numbers in the OC to an extent that maintains a proper cytoarchitectural framework, thus preventing further atrophy of the sensory epithelium.

Tissues samples for qRT-PCR were collected prior to noise exposure (pre-noise) or at 24 and 48 hr after the acoustic insult (post-noise). The noise exposure paradigm employed in this study resulted in significantly elevated cochlear Hes1 mRNA levels (∼1.8-fold at 24 hr and ∼1.5-fold at 48 hr after noise exposure; Figure 8 A), similar to previously published reports of noise-induced Notch pathway activation in guinea pigs.The noise exposure also resulted in significantly elevated Hes5 mRNA levels in the cochlea 48 hr after noise exposure (∼1.7-fold compared to pre-noise; p < 0.01) whereas only a modest elevation was observed at 24 hr after noise exposure (∼1.3-fold compared to pre-noise; p > 0.05).

(A) Hes1 mRNA expression in the cochlea becomes elevated in response to acute noise injury. The acoustic trauma employed in this study (125 dB SPL centered at 4 kHz for 3 hr) resulted in elevated cochlear Hes1 mRNA levels (∼1.8-fold at 24 hr and ∼1.5-fold at 48 hr after noise exposure; *p < 0.05 and ***p < 0.001 versus pre-noise control group; n = 3 guinea pigs per group). Tissue samples for qRT-PCR were either collected prior to noise exposure (pre-noise) or at 24 and 48 hr after the noise injury (post-noise). (B) Supporting cell quantification among sham- and siHes1-NP-treated ears of noise-deafened guinea pigs is shown. SCs were counted in the OHC regions corresponding to the 1-, 4-, 16-, and 32-kHz tonotopic frequency positions from age-matched undamaged controls (dotted line) or noise-injured cochleae treated with either scRNA (sham) NPs (solid line) or siHes1 NPs (gray line) and statistically analyzed. The number of SCs in noise-injured cochleae treated with scRNA NPs was significantly decreased in all four frequency regions compared to normal controls or the siHes1 NP treatment group (p < 0.01 or 0.001). Although reduced relative to undamaged controls, SC numbers in siHes1-NP-treated ears were markedly higher than sham-treated ears across the 4- to 32-kHz regions (###, all p < 0.001). Within the scRNA group, supporting cell numbers were significantly less at 16- and 32-kHz tonotopic positions compared to the number counted at the 1-kHz position (all p < 0.01). Errors bars represent the SEM. Numbers in parentheses represent the number of ears evaluated in each group. ** and *** indicate statistically lower SC counts compared to the normal control group (p < 0.01 and 0.001, respectively); ### indicates a statistically significant difference (p < 0.001) in SC numbers compared to the siHes1 NP treatment group.

A distinct population of hair-cell-like cells bearing dense bundles of short, disorganized stereocilia was also observed in the basal turns of cochleae treated with siHes1 NPs ( Figures 7 C–7E). These stereocilia were about 1 μm in length and were markedly taller and distinguishable from the ciliary structures on adjacent SCs, instead resembling the apical surfaces of immature HCs.Again, no such structures were observed among OCs treated with scRNA NPs ( Figures 7 A and 7B).

(C–E) Clusters of immature HC-like cells with dense and short stereocilia were also observed in the basal turn of cochleae adjacent to the pillar cell region at nine weeks after siHes1 NP treatment. (A and B) Such cells were not observed in noise-ablated cochleae treated with scRNA NPs. I, P, and O in (A) and (C) indicate IHCs, pillar cells, and OHCs, respectively. The scale bars represent 10 μm in (C) for (A) and (C) and 1 μm in (E) for (B), (D), and (E).

Clusters of Immature HCs with Short, Dense Stereocilia Bundles Were Observed by Scanning Electron Microscopy in Noise-Deafened Cochleae Treated with siHes1 NPs

Figure 7 Clusters of Immature HCs with Short, Dense Stereocilia Bundles Were Observed by Scanning Electron Microscopy in Noise-Deafened Cochleae Treated with siHes1 NPs

At both three and nine weeks after treatment, OHCs with short kinocilia and stereocilia were observed in cochleae treated with siHes1 NPs ( Figures 6 C–6F). Unlike the canonical v-shaped, stair-step-like stereocilia organization of mature OHCs, the stereocilia on these HCs were short, uniform in length, and angled toward the center of the bundle. The kinocilia on these HCs were approximately 2.5 μm in length ( Figures 6 E and 6F), whereas kinocilia on mature HCs in undamaged cochleae are approximately 8 μm in length.No such scanning electron microscope evidence for immature HCs was observed in scRNA-NP-treated OCs, where the majority of OHCs were eliminated ( Figures 6 A and 6B).

(A and B) OHCs were efficiently ablated in the basal turns of noise-injured OCs treated with scRNA NPs at both 3 (A) and 9 (B) weeks after treatment. The original OHC region appeared narrow and progressively diminished over time (A and B). (C and D) In contrast, noise-injured ears treated with siHes1 NPs presented with a significant population of basal OHCs, the majority of which were in cytoarchitecturally correct positions within the OC and possessed morphologically mature stereociliary structures. However, HCs bearing short stereocilia, which angled toward the center of the bundle and lacked the canonical stair-step organization, were also uniquely observed in the OHC region of OCs treated with siHes1 NPs (C–F). I, P, and O in (A)–(D) indicate IHCs, pillar cells, and OHCs, respectively. The scale bars represent 10 μm in (D) for (A)–(D) and 1 μm in (E) and (F).

Prestin and vGluT3 are terminal differentiation markers for OHCs and IHCs, respectively, and can thus be used to distinguish mature from immature HCs in the OC.Although the vast majority of repopulated IHCs and OHCs at both intermediate and terminal evaluation intervals (i.e., three and nine weeks post-treatment) appeared phenotypically normal, immature myosin-VIIa-positive OHCs, lacking prestin expression, were also observed in OCs treated with siHes1 NPs at three weeks after treatment ( Figure 4 ). Whereas some of these immature, prestin-negative OHCs possessed normal cellular morphology ( Figure 4 H’), others presented with degenerative features, such as condensed or fragmented nuclei and shrunken cell bodies ( Figure 4 I’).Immature IHCs immunopositive for myosin VIIa but lacking detectable vGluT3 expression were also uniquely observed in cochleae treated with siHes1 NPs ( Figure 5 ). No such immature myosin-VIIa-positive/prestin-negative OHCs or myosin-VIIa-positive/vGluT3-negative IHCs were observed in OCs from cochleae treated with scRNA NPs ( Figures 4 and 5 ).

(A–F) HCs were immunolabeled with myosin VIIa antibodies (green in A, B, G, and H), whereas mature IHCs were differentially immunolabeled with vGluT3 (red in C, D, G, and H). Nuclei were labeled with DAPI (blue in E–H). Arrows in (B) and (H) indicate a myosin-VIIa-positive/vGluT3-negative ectopic immature IHC in a noise-injured OC at nine weeks post-siHes1 NP treatment. Arrowheads in (B), (D), and (H) indicate a myosin VIIa/vGluT3-positive ectopic IHC in the same siHes1-NP-treated OC. All IHCs in OCs treated with scRNA NPS were double labeled with myosin VIIa and vGluT3 (G). The scale bar represents 10 μm in (H) for (A)–(H).

Immature OHCs were observed in OCs treated with siHes1 NPs by differential prestin immunolabeling at nine (A–H’) and three (I and I’) weeks after treatment. HCs were immunolabeled by anti-myosin VIIa (green in A, B, B’, and G–I’), whereas mature OHCs were immunolabeled with anti-prestin (pink in C–D’ and G–I’). Nuclei were labeled with DAPI (blue in E–I’). Arrowheads in B, D, and H indicate a myosin-VIIa-positive/prestin-negative immature OHC in the 2 nd turn of a noise-injured OC treated with siHes1 NPs at nine weeks post-treatment. This immature OHC has an apparently normal OHC morphology (H’). Arrowheads in (I) indicate two myosin-VIIa-positive/prestin-negative OHCs adjacent to a myosin-VIIa-positive/prestin-positive OHC in a noise-injured OC at three weeks post-siHes1 NP treatment. These OHCs were anucleate and small in size (I’). All OHCs in noise-deafened ears treated with scRNA NPs possessed dual labeling with myosin Vlla and prestin (G). The scale bars represent 10 μm in (I) and (I’) and apply to (A)–(I) and (B’), (D’), (F’), (H’), and (I’), respectively.

Overactivation of Notch1 signaling induces ectopic hair cells in the mouse inner ear in an age-dependent manner.

Clusters of supernumerary IHCs were uniquely observed in 50% of the cochleae (5 out of 10) treated with siHes1 NPs at both three and nine weeks after treatment ( Figure 3 ). Whereas many of these ectopic, myosin VIIa-positive IHCs presented with phalloidin-positive stereociliary structures (arrowheads, Figures 3 D and 3F), some lacked either stereocilia or cuticular plates, indicative of a functionally immature morphology (arrows, Figures 3 B and 3F). Most of these ectopic HCs were located within the basal and second turns of the OC, proximal to the therapeutic infusion site ( Figure 3 ). No such ectopic HCs were observed in any of the noise-injured cochleae treated with scRNA NPs (0 out 8; Figures 3 A, 3C, and 3E).

(A–F) HCs were immunolabeled with anti-myosin VIIa (green in A, B, E, and F), whereas stereocilia were labeled with fluorophore-conjugated phalloidin (yellow in C–F). Nuclei were stained with DAPI (blue in E and F). Supernumerary IHCs (arrows in B and F) were observed only in noise-deafened OCs treated with siHes1 NPs. Some ectopic IHCs possessed phalloidin-labeled stereocilia (arrowheads in D and F), whereas some presented with no stereocilia (arrows in F). No ectopic HCs were observed in cochleae treated with scRNA NPs (A and E). (G and H) Scanning electron microscope image of an ectopic IHC in an siHes1-NP-treated OC is shown. Ectopic IHCs (arrow in G) with stereociliary structures were also observed by scanning electron microscopy in OCs at three weeks after siHes1 NP treatment (image collected from the 2 nd turn of the OC). At this time point, the majority of HCs possessed stereocilia with normal morphology (G). I, P, and O in (G) indicate IHCs, pillar cells, and OHCs, respectively. The scale bars represent 50 μm in (F) for (A)–(F), 10 μm in (G), and 1 μm in (H).

The correlation between ABR thresholds and HC loss was analyzed at the 2-, 4-, 8-, and 16-kHz tonotopic frequency regions. A moderate to strong correlation was observed between ABR thresholds and OHC loss, as well as between ABR thresholds and IHC loss (*p < 0.05 and **p < 0.01; Table 1 ). In general, a stronger correlation was observed in the lower (2 and 4 kHz) frequency regions than in the higher (8 and 16 kHz) frequency regions ( Table 1 ). These results are consistent with previous reports.

Quantitative evaluation of IHC numbers in siHes1-NP-treated ears also demonstrated an overall treatment-specific restoration relative to sham-treated controls (54.3% missing IHCs in the scRNA group compared to 24.9% in the siHes1 groups; p < 0.001; Figure 2 D). As was observed for OHC recovery, the high-frequency region in this cohort presented with greater net restoration of IHC numbers (Δ45.1% or approximately 370 IHCs/cochlea) than the low-frequency region (Δ18.5% or approximately 270 IHCs/cochlea; p < 0.001). However, the relative extent of IHC restoration (∼2-fold) between these two regions of the OC was similar, as the extent of IHC loss in the low-frequency region was approximately 50% less than that observed in the high-frequency region. As denoted in the previous section, the injurious noise exposure was centered at a frequency of 4 kHz ( Figures 2 A and 2B), suggesting that the excitotoxic trauma around this tonotopic position was anticipated to be the most severe.The results from these quantitative HC evaluations support this hypothesis, as both greater HC losses and lesser therapeutic restoration were contextually observed across the 2.7–5.4 kHz tonotopic range in scRNA- and siHes1-NP-treated ears, respectively.

Changing relationships between structure and function in the cochlea during recovery from intense sound exposure.

In noise-deafened cochleae treated with siHes1 NPs, the average percentage of missing OHCs and IHCs (55.9% and 24.9%, respectively) at nine weeks post-treatment was significantly reduced across the entire span of the OC (all p < 0.001 compared to the scRNA-NP-treated group; Figures 2 A and 2B). Regionalized siHes1-NP-specific HC restoration was statistically analyzed and is graphically depicted in Figures 2 C and 2D. From this analysis, statistically significant increases in average OHC numbers (Δ27.2% or approximately 1,960 OHCs/cochlea) were observed across all (low + high) tonotopic positions in siHes1-NP-infused cochleae relative to scRNA NP controls. Although the high-frequency region (4.9–54.7 kHz) of the OC presented with a greater overall degree (p < 0.001) of treatment-specific OHC restoration (Δ37.8% or approximately 1,123 OHCs/cochlea) than the more apical, low-frequency region (0.1–4.6 kHz, Δ19.7%, or approximately 835 OHCs/cochlea), both areas experienced similar relative gains in total OHCs (∼1.6-fold versus ∼1.4-fold in high- and low-frequency regions, respectively; Figure 2 C).

HC counts were conducted along the basilar membranes among cochlea harvested from the scRNA NP and siHes1 NP cohorts at nine weeks post-treatment. Cytocochleograms showing the average percentage of missing myosin VIIa-positive outer HCs (OHCs) and inner HCs (IHCs) between the two treatment groups are depicted in Figures 2 A and 2B, respectively. As predicted, the noise injury caused pervasive HC loss throughout the cochlea. In sham (scRNA)-NP-treated ears, the majority of OHCs (98.9%) and IHCs (83.2%) were completely ablated in the basal and second turns (tonotopic frequency ranges of 4.9–54.7 kHz) of the OC, whereas the HC quantification across lower tonotopic frequency positions (0.1–4.6 kHz) revealed a reduced, albeit pronounced, incidence of OHC (72.0%) and IHC (34.1%) loss. In total, the average percentages of missing OHCs and IHCs in scRNA-treated cochleae were 83.1% and 54.3%, respectively.

Average post-deafening ABR threshold recoveries (Δ dB) were calculated by subtracting ABR threshold recovery (i.e., [ABR threshold at 24 hr after noise exposure] − [ABR threshold at 9 weeks after treatment]) in the scRNA-NP-treated ears from ABR threshold recovery in the siHes1-NP-treated ears at each frequency at the terminal, nine-week, post-treatment sampling interval. From this analysis, the average threshold recovery across all four test frequencies in the siHes1-NP-treatment group was 14.64 dB SPL, whereas a recovery of only 2.71 dB SPL was achieved in the scRNA group (p < 0.001). In the siHes1-NP-treated group, average threshold recoveries exhibited a basal-to-apical gradient of improvement, such that, at 2, 4, 8, and 16 kHz, the threshold recoveries were 9.29, 11.43, 17.14, and 20.71 dB SPL, respectively. No such gradient effect was observed in the sham (scRNA)-NP-treated cohort, in which the corresponding average frequency-dependent recoveries in treated ears were −3.33, 0, 6.67, and 7.5 dB. These results translated to statistically significant siHes1-NP-specific hearing recovery at each test frequency, with an average improvement of greater than 10 dB relative to scRNA NP control-treated ears (p < 0.05 or 0.01; Figure 1 D).

In contrast to the scRNA-NP-treated cohort, statistically significant hearing recovery was observed among ears infused with siHes1 NPs across test frequencies of 4–16 kHz, beginning at three weeks post-treatment and extending out to the terminal sampling interval of nine weeks post-treatment (p < 0.05, 0.01, or 0.001; Figure 1 C). Although the in-group hearing recovery at 2 kHz was not calculated to be statistically significant, an average ABR threshold improvement of 5.7 dB SPL was measured at this frequency at three weeks post-treatment, which increased to 9.3 dB SPL at the terminal nine-week recording interval, indicative of ongoing functional recovery at this tonotopic position. Modest progressive recovery was also measured across the experimental time course at 8 kHz in the siHes1-NP-treated cohort, whereas maximal hearing recovery at the 4- and 16-kHz test frequencies were achieved at three weeks post-treatment ( Figure 1 C). It should be noted that, based on the parameters of our noise-exposure paradigm, the 4-kHz tonotopic region of the cochlea likely served as the epicenter of trauma (see Figures 1 B and 2 ).

(A and B) The average number of missing OHCs (A) and IHCs (B) along the length of the basilar membrane in surface preparations of cochleae collected at 9 weeks after scRNA or siHes1 NP treatment is depicted as cytocochleograms. Shaded areas in (A) and (B) indicate the frequency range of the 4-kHz octave band noise exposure. Almost 100% of OHCs were eliminated in the tonotopic frequency positions ranging from 4.9 to 54.7 kHz in the scRNA group (A), whereas approximately 80% of IHCs were eliminated across this frequency region (B). Significantly less overall HC loss was observed in the siHes1 NP treatment group compared to the scRNA treatment group (***p < 0.001 in A and B). (C and D) IHC (C) and OHC (D) recovery in the low-frequency region (low in C and D; 0.1–4.6 kHz), the high-frequency region (high in C and D; 4.9–54.7 kHz), and among all frequency regions (all in C and D; 0.1–54.7 kHz) were calculated and statistically analyzed. Significant OHC and IHC recovery was observed in the siHes1 NP treatment group across all tonotopic frequency regions and within each of the two frequency subgroups compared to the scRNA group (**p < 0.01 and ***p < 0.001). Δ dB indicates HC recovery in the siHes1 NP group compared to the scRNA NP group within each frequency range. Numbers in parentheses represent the number of ears evaluated in each group. Errors bars represent SEM.

Changing relationships between structure and function in the cochlea during recovery from intense sound exposure.

Sham (scRNA) or therapeutic (siHes1) NP treatment was initiated via unilateral intracochlear infusion by mini-osmotic pumps at 72 hr post-deafening. Twenty-four hours later, the treatment was terminated upon surgical extraction of the pumps. Serial ABR threshold measurements were conducted at three, six, and nine weeks after treatment in all animals. In the scRNA-NP-treated ears, thresholds remained above 85 dB SPL at all test frequencies at three weeks post-treatment. At subsequent test intervals of six and nine weeks post-treatment, marginally lower thresholds (4 or 5 dB) were recorded at 8 and 16 kHz, whereas no improvements in auditory function were recorded at 2 and 4 kHz ( Figure 1 B; all p > 0.05). These results revealed that no statistically significant hearing recovery occurred in the sham-NP-treated ears, indicating that the acoustic trauma induced both profound and permanent deafness.

Prior to the deafening noise exposure, the young adult guinea pigs employed in this study exhibited normal baseline auditory brainstem response (ABR) thresholds of 5–30 dB sound press level (SPL) at the 2-, 4-, 8-, and 16-kHz test frequencies (all p > 0.05). However, due to large inter-animal variability to noise exposure in guinea pigs,some guinea pigs did not exhibit sufficient hearing loss at 24 hr after the acoustic trauma as measured by ABR. To limit the influence of this inter-animal variation on the objective assessment of hearing recovery after treatment, animals that did not present with bilateral ABR thresholds greater than 80 dB SPL at any frequency at 24 hr after noise exposure (125 dB SPL centered at 4 kHz for three hours) were excluded from the study. After application of this exclusion criterion, deafened guinea pigs were randomly assigned to one of two cohorts designated for either sham (scrambled siRNA [scRNA]) or Hes1 siRNA (siHes1) NP treatment. The average pre-treatment threshold shifts observed in the two groups for each test frequency were statistically identical, except at 2 kHz, where significantly greater hearing loss was observed in the siHes1 NP treatment group (p < 0.01; Figure 1 A).

(A) Pre-treatment hearing threshold shifts in the scRNA NP and siHes1 NP cohorts at 24 hr after a deafening noise exposure (125 dB SPL centered at 4 kHz for 3 hr). Statistically identical threshold shifts at each test frequency were recorded in the two groups, except at 2 kHz, where significantly greater hearing loss was observed in the siHes1 NP cohort (**p < 0.01). (B) No significant hearing improvement was observed in noise-deafened guinea pigs treated with scRNA NPs at any time point after treatment (all p > 0.05). (C) In comparison to pre-treatment ABR thresholds, significant hearing improvement was observed at all time points (3, 6, and 9 weeks after treatment) in siHes1-NP-treated ears across the 4- to 16-kHz frequency range. ***p < 0.001 versus 3 weeks after treatment; ###p < 0.001 versus 6 weeks after treatment; &p < 0.05 and &&&p <0.001 versus 9 weeks after treatment. (D) Significant hearing improvement, as measured by ABR threshold recovery, was observed in the siHes1 NP treatment group at all frequencies tested compared to the scRNA NP treatment group (*p < 0.05 and **p < 0.01). Δ dB indicates frequency-specific hearing improvement in the siHes1 NP treatment group compared to the scRNA NP treatment group. Numbers in parentheses represent the number of ears evaluated in each group. Errors bars represent SEM.

Discussion

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Ferekidis E. Intratympanic steroid treatment in idiopathic sudden sensorineural hearing loss: a control study. Consistent with our findings, pharmacologic manipulation of the Notch pathway has previously been shown to restore cochlear HC numbers and a degree of hearing recovery in adult animals after a deafening acoustic trauma,despite the fact that previous studies had suggested that the capacity for cochlear HC regeneration through attenuation of Notch signaling declines considerably during developmental maturation of the OC.However, the magnitude and tonotopic breadth of functional restoration reported herein for targeted silencing of a single Notch effector gene was seemingly more robust than that anticipated from previous studies, with more than 10 dB of ABR threshold recovery measured at test frequencies of 4, 8, and 16 kHz in siHes1-NP-treated ears and greater than 10 dB improvement in all four test frequencies, including 2 kHz, in comparison to scRNA-NP-treated controls. In a series of published studies on speech intelligibility in humans, it has been consistently determined that the just-noticeable-difference (JND) in discriminating a difference in sound intensity lies between 2 and 3 dB.Therefore, a 3-dB improvement in hearing threshold would be clinically relevant, whereas a 6-dB improvement would represent a doubling of the JND. The measured threshold improvements of 10 dB or more reported herein at multiple test frequencies represent more than a 3× JND improvement and a greater than two-fold increase in perceived loudness, which would be predicted to correspond to clinically detectable differences in speech threshold in humans.Moreover, a 10-dB improvement in hearing function at one or more frequencies has been used as a primary endpoint criterion for assessing clinically meaningful hearing improvement in human clinical studies.

37 Atkinson P.J.

Wise A.K.

Flynn B.O.

Nayagam B.A.

Richardson R.T. Hair cell regeneration after ATOH1 gene therapy in the cochlea of profoundly deaf adult guinea pigs. , 53 Liu Z.

Dearman J.A.

Cox B.C.

Walters B.J.

Zhang L.

Ayrault O.

Zindy F.

Gan L.

Roussel M.F.

Zuo J. Age-dependent in vivo conversion of mouse cochlear pillar and Deiters’ cells to immature hair cells by Atoh1 ectopic expression. 53 Liu Z.

Dearman J.A.

Cox B.C.

Walters B.J.

Zhang L.

Ayrault O.

Zindy F.

Gan L.

Roussel M.F.

Zuo J. Age-dependent in vivo conversion of mouse cochlear pillar and Deiters’ cells to immature hair cells by Atoh1 ectopic expression. , 54 Costa A.

Sanchez-Guardado L.

Juniat S.

Gale J.E.

Daudet N.

Henrique D. Generation of sensory hair cells by genetic programming with a combination of transcription factors. , 55 Xiang M.

Gao W.Q.

Hasson T.

Shin J.J. Requirement for Brn-3c in maturation and survival, but not in fate determination of inner ear hair cells. In addition to being robust, the treatment-specific functional recovery elicited by cochlear siHes1 NP infusion was also enduring. Several HC regenerative studies have documented treatment-specific restoration of transient populations of HC-like cells in the OC that remain morphologically and functionally immature and are subsequently lost.Our results demonstrate the restoration of sustainable IHC and OHC populations throughout the OC and an accompanying functional recovery that persisted from the initial ABR recording interval at three weeks post-treatment and extended out to the terminal nine-week interval. However, we identified subpopulations of immature, myosin-VIIa-positive OHC-like cells and IHC-like cells that were either prestin- or vGluT3-negative, respectively, or possessed rudimentary stereocilia bundles, indicating that some new HCs failed to achieve functional maturity over the time course of this study. Additionally, anucleate, degenerating myosin-VIIa-positive/prestin-negative OHCs were also observed at three weeks post-treatment, consistent with a degree of deterioration among a subpopulation of the newly repopulated HCs (arrowheads in Figures 4 I and 4I’). These observations may indicate that additional developmental factors or cellular constituents may be contextually required for optimal realization of the mature phenotype.Nonetheless, a significant proportion of the repopulated IHCs and OHCs that were observed in the siHes1-NP-treatment group presented with biomarkers and stereociliary structures that reflected functional maturation.

32 Bohne B.A.

Kimlinger M.

Harding G.W. Time course of organ of Corti degeneration after noise exposure. , 56 Harding G.W.

Bohne B.A. Noise-induced hair-cell loss and total exposure energy: analysis of a large data set. 57 Wang J.

Ruel J.

Ladrech S.

Bonny C.

van de Water T.R.

Puel J.-L. Inhibition of the c-Jun N-terminal kinase-mediated mitochondrial cell death pathway restores auditory function in sound-exposed animals. 20 Mizutari K.

Fujioka M.

Hosoya M.

Bramhall N.

Okano H.J.

Okano H.

Edge A.S. Notch inhibition induces cochlear hair cell regeneration and recovery of hearing after acoustic trauma. 58 Coleman J.K.M.

Kopke R.D.

Liu J.

Ge X.

Harper E.A.

Jones G.E.

Cater T.L.

Jackson R.L. Pharmacological rescue of noise induced hearing loss using N-acetylcysteine and acetyl-L-carnitine. Although we are unable to formally rule out the possibility that siHes1 NP treatment may confer a degree of protection against the loss of pre-existing HCs in noise-injured ears in this non-transgenic animal model, several lines of evidence suggest otherwise. For instance, previous studies have demonstrated that HC loss in mammals occurs rapidly (within the first 12 hr post-injury) following a prolonged high-energy noise exposure.In related studies, approximately 65% of HCs were ablated in guinea pig cochleae within the first 48 hr after a 130-dB SPL noise exposure for 15 min, and the percentage reached 85% at 5 days after noise exposure.We exposed the animals to 125 dB SPL noise for 3 hr, the sound energy of which is 3.79 times of a 130-dB SPL noise exposure for 15 min ( http://www.eurovent-certification.com/fic_bdd/pdf_fr_fichier/1137149375_Review_67-Bill_Cory.pdf ), suggesting a far greater damaging effect on primary HC loss. Using a transgenic mouse model to lineage trace the origins of new HCs, Mizutari and colleagues demonstrated that regenerative intervention at 24 hr post-injury using a gamma-secretase inhibitor (GSI) for pan Notch inhibition did not result in significant preservation of pre-existing HCs, indicating that an even more acute inhibition of Notch and Hes1 signaling was not protective against HC loss.Moreover, other effective intervention strategies, such as antioxidant intervention with N-acetylcysteine, that are designed to mitigate the progressive pathophysiological responses induced by an acute acoustic trauma (AAT) have been shown to be ineffective when administered more than one hour post-injury.Therefore, in light of the fact that we delayed intervention for 72 hr post-trauma, the most plausible explanation for the degree and tonotopic breadth of HC restoration that we observed in siHes1-NP-treated ears is that of bona fide regeneration.

20 Mizutari K.

Fujioka M.

Hosoya M.

Bramhall N.

Okano H.J.

Okano H.

Edge A.S. Notch inhibition induces cochlear hair cell regeneration and recovery of hearing after acoustic trauma. , 23 Du X.

Li W.

Gao X.

West M.B.

Saltzman W.M.

Cheng C.J.

Stewart C.

Zheng J.

Cheng W.

Kopke R.D. Regeneration of mammalian cochlear and vestibular hair cells through Hes1/Hes5 modulation with siRNA. Generation of new HCs from direct transdifferentiation of SCs is predicted to occur at the expense of the SC population, thus reducing their prevalence in OCs.Consistent with this rationale, we observed a decline in the number of DAPI-stained nuclei in the SC layer (relative to undamaged control ears) in siHes1-NP-treated ears across the 1-, 4-, 16-, and 32-kHz tonotopic frequency regions sampled in our analyses (297.5, 295, 290, and 198.33 SCs/mm for 1, 4, 16, and 32 kHz, respectively; Figure 8 B). However, these apparent SC deficits were only sufficient to account for the extent of repopulated HCs in the 32-kHz tonotopic region among siHes1-NP-treated ears. Interpretation of these results is complicated by the fact that markedly greater SC loss was observed in scRNA-NP-treated ears, in which widespread HC restoration was not observed.

18 Izumikawa M.

Minoda R.

Kawamoto K.

Abrashkin K.A.

Swiderski D.L.

Dolan D.F.

Brough D.E.

Raphael Y. Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. 18 Izumikawa M.

Minoda R.

Kawamoto K.

Abrashkin K.A.

Swiderski D.L.

Dolan D.F.

Brough D.E.

Raphael Y. Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. 59 Woods C.

Montcouquiol M.

Kelley M.W. Math1 regulates development of the sensory epithelium in the mammalian cochlea. , 60 White P.M.

Doetzlhofer A.

Lee Y.S.

Groves A.K.

Segil N. Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells. , 61 Li H.

Liu H.

Heller S. Pluripotent stem cells from the adult mouse inner ear. 62 Li W.

Wu J.

Yang J.

Sun S.

Chai R.

Chen Z.-Y.

Li H. Notch inhibition induces mitotically generated hair cells in mammalian cochleae via activating the Wnt pathway. 63 Cotanche D.A.

Kaiser C.L. Hair cell fate decisions in cochlear development and regeneration. 32 Bohne B.A.

Kimlinger M.

Harding G.W. Time course of organ of Corti degeneration after noise exposure. Several explanations can be proffered to account for these apparent discrepancies. For instance, a similar finding was previously documented by Izumikawa and colleagues following adenoviral-mediated SC transdifferentiation through forced Atoh1 expression in noise-deafened guinea pigs.In these animals, the active therapeutic paradoxically increased the number of SCs in the OCs after treatment relative to the widespread losses documented in untreated ears.The authors postulated that non-sensory cells may have been recruited to surround new HCs to structurally, if not functionally, replace transdifferentiated SCs through migratory and/or proliferative processes.Although forced transdifferentiation through inhibition of Notch signaling has previously been thought to represent a non-mitotic process in mammalian cochleae, recent work by Li and colleagues has challenged that paradigm by demonstrating that contextual in vivo deletion of Notch1 in post-natal OCs was capable of inducing a significant SC proliferative response.Thus, it is possible that reversible silencing of the Notch pathway effector, Hes1, may elicit a similar response in vivo, thereby promoting a degree of in situ SC replacement in a manner analogous to that which occurs in avian cochleae.In scRNA-NP-treated cochleae, the marked SC loss that we observed at nine weeks post-injury is most likely attributable to progressive degeneration of the OC following excitotoxic loss of HCs, particularly in the basal turn. Indeed, in this region of the OC, flat epithelium was evident by light microscopy and scanning electron microscopy in scRNA-NP-treated ears. A similar profile of delayed, yet pervasive, SC loss in response to a high-energy acoustic trauma that resulted in the widespread “wipe out” of HCs was recently documented in chinchillas, coinciding with the formation of flat epithelium.Thus, our observations on the effects of trauma and treatment on SC populations within the OC indicate that, in the absence of HC restoration and the appropriate cellular microenvironment, the residual SC population experienced widespread apoptosis and conversion to an expanded squamous epithelium.

23 Du X.

Li W.

Gao X.

West M.B.

Saltzman W.M.

Cheng C.J.

Stewart C.

Zheng J.

Cheng W.

Kopke R.D. Regeneration of mammalian cochlear and vestibular hair cells through Hes1/Hes5 modulation with siRNA. , 64 Jawahar N.

Meyyanathan S. Polymeric nanoparticles for drug delivery and targeting: a comprehensive review. , 65 Mudshinge S.R.

Deore A.B.

Patil S.

Bhalgat C.M. Nanoparticles: emerging carriers for drug delivery. , 66 Woodrow K.A.

Cu Y.

Booth C.J.

Saucier-Sawyer J.K.

Wood M.J.

Saltzman W.M. Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA. 67 Mohanraj V.J.

Chen Y. Nanoparticles – a review. , 68 Vauthier C.

Bouchemal K. Methods for the preparation and manufacture of polymeric nanoparticles. , 69 Yameen B.

Choi W.I.

Vilos C.

Swami A.

Shi J.

Farokhzad O.C. Insight into nanoparticle cellular uptake and intracellular targeting. 23 Du X.

Li W.

Gao X.

West M.B.

Saltzman W.M.

Cheng C.J.

Stewart C.

Zheng J.

Cheng W.

Kopke R.D. Regeneration of mammalian cochlear and vestibular hair cells through Hes1/Hes5 modulation with siRNA. , 70 Cai H.

Wen X.

Wen L.

Tirelli N.

Zhang X.

Zhang Y.

Su H.

Yang F.

Chen G. Enhanced local bioavailability of single or compound drugs delivery to the inner ear through application of PLGA nanoparticles via round window administration. , 71 Ge X.

Jackson R.L.

Liu J.

Harper E.A.

Hoffer M.E.

Wassel R.A.

Dormer K.J.

Kopke R.D.

Balough B.J. Distribution of PLGA nanoparticles in chinchilla cochleae. , 72 Tamura T.

Kita T.

Nakagawa T.

Endo T.

Kim T.-S.

Ishihara T.

Mizushima Y.

Higaki M.

Ito J. Drug delivery to the cochlea using PLGA nanoparticles. , 73 Youm I.

Musazzi U.M.

Gratton M.A.

Murowchick J.B.

Youan B.C. Label-free ferrocene-loaded nanocarrier engineering for in vivo cochlear drug delivery and imaging. , 74 Youm I.

West M.B.

Li W.

Du X.

Ewert D.L.

Kopke R.D. siRNA-loaded biodegradable nanocarriers for therapeutic MAPK1 silencing against cisplatin-induced ototoxicity. 23 Du X.

Li W.

Gao X.

West M.B.

Saltzman W.M.

Cheng C.J.

Stewart C.

Zheng J.

Cheng W.

Kopke R.D. Regeneration of mammalian cochlear and vestibular hair cells through Hes1/Hes5 modulation with siRNA. , 75 Panyam J.

Zhou W.-Z.

Prabha S.

Sahoo S.K.

Labhasetwar V. Rapid endo-lysosomal escape of poly(DL-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery. We used PLGA NPs as carriers to deliver siRNA into the cellular constituents of the cochlea. The technological advantages of using NPs as drug carriers are myriad, including enhanced stability, high carrier capacity, sustained release, and biocompatibility with sensitive epithelia.PLGA encapsulation is also advantageous over naked RNA, as it allows for tailored optimization and strategic evolution of the therapeutic formulation, such that siRNA release rates and particle degradation characteristics can be fine-tuned by modifying the properties of the matrix constituents, and therapeutic payloads can be strategically targeted to desired cellular or tissue components by introducing homing factors (e.g., ligands or antibodies) to the NP surface.Our lab and others have shown that PLGA-based NPs efficiently cross cell boundaries and are efficiently endocytosed by a variety of cells within the cochleae, including SCs.Once internalized, the intrinsic capacity of the PLGA NP surface to become reversibly cationized in the low pH environment of endo-lysomal vesicles allows for the rapid (<10 min) release of these delivery agents into the cytoplasm, preventing the hydrolytic degradation of the unmodified siRNA payloads within this intracellular compartment. Upon cytoplasmic release, progressive erosion of the PLGA matrix promotes sustained silencing of target genes through continuous release of the siRNA payload, as has been previously demonstrated in cochlear tissues for siHes1.These inherent attributes provide a compelling foundation for further refinement of this therapeutic approach that leaves the established efficacy of the therapeutic payload unchanged while potentially enhancing the efficiency and specificity of drug delivery.