Figure - available via license: Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported
Content may be subject to copyright.
Crista ampullaris transfected with Cy3-labeled siRNA (red) and stained with Alexa 488 Phalloidin (green) and To-Pro-3 (blue). The shown siRNAs were successfully delivered into hair cells of the crista ampullaris with TAT-DRBD after 24 h (a), and siRNA uptake was greater in the dark cells on the planum semilunatum at the bottom of the crista ampullaris. Strong red fluorescence was still present after 72 h (b), and siRNA uptake by dark cells had not yet been eliminated after 7 days (c). In control ears, no siRNA was found after round-window diffusion at 24 h, 72 h and 7 days (d–f); ‘⋆' show the red fluorescence of Cy3 was only found on membrane of hair cells but could not be found in the cytoplasm.
Source publication
The use of small-interfering RNA (siRNA) has great potential for the development of drugs designed to knock down the expression of damage- or disease-causing genes. However, because of the high molecular weight and negative charge of siRNA, it is restricted from crossing the blood-cochlear barrier, which limits the concentration and size of molecul...
Citations
... While these in vitro results are promising, in vivo confirmation in animal models is a necessary next step to replicate the complex physiological environment of the mammalian cochlea and is desired for future drug screening. In future studies, it will be worthwhile to investigate the efficacy of pharmacological inhibitors or RNAi-based approaches to block the functions of these proteins, as has been demonstrated with other targets [67][68][69][70][71][72][73]. ...
Given the non-labile, terminal differentiation of inner-ear sensory cells, preserving their function is critical since sensory cell damage results in irreversible hearing loss. Gentamicin-induced cytotoxicity is one of the major causes of sensory cell damage and consequent sensorineural hearing loss. However, the precise molecular mechanisms and target proteins involved in ototoxicity are still unknown. The objective of the present study was to identify target proteins involved in gentamicin-induced cytotoxicity to better characterize the molecular pathways involved in sensory cell damage following ototoxic drug administration using House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). We identified several unique proteins involved in gentamicin-induced cytotoxicity, expression of which were further confirmed using confocal microscopy. Further investigation of these pathways can inform the design and discovery of novel treatment modalities to prevent sensory cell damage and preserve their function.
... Drugs can also be targeted to specific regions of the cochlea using repeat injection strategies and controlled-release systems, such as biodegradable biopolymers and catheters (Salt and Plontke, 2009). Multiple studies have used the intratympanic approach to deliver steroids, proteins, and viral vectors to the middle ear and cochlea (Qi et al., 2014;Sayoo and Kumar, 2019;Yu et al., 2012). Additionally, ASOs of varying chemical composition were shown to reach auditory hair cells and fibrocytes of the mammalian cochlea upon intratympanic delivery (Delprat et al., 2002;Lentz et al., 2020). ...
Hearing loss affects more than 430 million people, worldwide, and is the third most common chronic physical condition in the United States and Europe (GBD Hearing Loss Collaborators, 2021; NIOSH, 2021; WHO, 2021). The loss of hearing significantly impacts motor and cognitive development, communication, education, employment, and overall quality of life. The inner ear houses the sensory organs for both hearing and balance and provides an accessible target for therapeutic delivery. Antisense oligonucleotides (ASOs) use various mechanisms to manipulate gene expression and can be tailor-made to treat disorders with defined genetic targets. In this review, we discuss the preclinical advancements within the field of the highly promising ASO-based therapies for hereditary hearing loss disorders. Particular focus is on ASO mechanisms of action, preclinical studies on ASO treatments of hearing loss, timing of therapeutic intervention, and delivery routes to the inner ear.
... induces sequence-specific degradation of targeted mRNA and can specifically inhibit the replication of influenza virus [39]. In general, siRNA cleavage action depends on its specific features [40]. The siRNA tools have been widely used in the research of anti-influenza viral infection, because they have high specificity and selectivity as well as silencing efficiency. ...
Influenza is an important research subject around the world because of its threat to humanity. Influenza A virus (IAV) causes seasonal epidemics and sporadic, but dangerous pandemics. A rapid antigen changes and recombination of the viral RNA genome contribute to the reduced effectiveness of vaccination and anti-influenza drugs. Hence, there is a necessity to develop new antiviral drugs and strategies to limit the influenza spread. IAV is a single-stranded negative sense RNA virus with a genome (viral RNA—vRNA) consisting of eight segments. Segments within influenza virion are assembled into viral ribonucleoprotein (vRNP) complexes that are independent transcription-replication units. Each step in the influenza life cycle is regulated by the RNA and is dependent on its interplay and dynamics. Therefore, viral RNA can be a proper target to design novel therapeutics. Here, we briefly described examples of anti-influenza strategies based on the antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA) and catalytic nucleic acids. In particular we focused on the vRNA structure-function relationship as well as presented the advantages of using secondary structure information in predicting therapeutic targets and the potential future of this field.
... Cell-penetrating peptides are composed of 6 to 30 amino acid residues able to increase cellular internalization and penetration in physiological barriers such as the blood-brain barrier (Silva et al., 2019). In preclinical studies, cell-penetrating peptides promote siRNA (Qi et al., 2014) and protein 770 (Kashio et al., 2012;Takeda et al., 2016) delivery to the inner ear. Thus, combining cell-penetrating peptides with nanocarriers, either linked to their surface (Yoon et al., 2015) or mixed with the nanocarrier suspension (Cai et al., 2017), should enhance the passage of nanocarriers through the round window membrane. ...
Despite the high incidence of inner ear disorders, there are still no dedicated medications on the market. Drugs are currently administered by the intratympanic route, the safest way to maximize drug concentration in the inner ear. Nevertheless, therapeutic doses are ensured for only a few minutes/hours using drug solutions or suspensions. The passage through the middle ear barrier strongly depends on drug physicochemical characteristics. For the past 15 years, drug encapsulation into nanocarriers has been developed to overcome this drawback. Nanocarriers are well known to sustain drug release and protect it from degradation. In this review, in vivo studies are detailed concerning nanocarrier biodistribution, their pathway mechanisms in the inner ear and the resulting drug pharmacokinetics. Key parameters influencing nanocarrier biodistribution are identified and discussed: nanocarrier size, concentration, surface composition and shape. Recent advanced strategies that combine nanocarriers with hydrogels, specific tissue targeting or modification of the round window permeability (cell-penetrating peptide, magnetic delivery) are explored. Most of the nanocarriers appear to be safe for the inner ear and provide a significant efficacy over classic formulations in animal models. However, many challenges remain to be overcome for future clinical applications.
... For the small interfering RNA (siRNA) transfection, mice were anesthetized, a 5 μl solution of Gja1 Silencer ® Select Pre-designed siRNA (40 μM, 4390771, Assay ID: s66667, Thermo Fisher Scientific, Waltham, MA) was injected through the posterior-inferior quadrant (Qi et al., 2014) with a Hamilton's microliter syringe (30 G needle), and the middle ear was filled completely with the solution for 3 days. Scrambled siRNA (4390846, Thermo Fisher Scientific, Waltham, MA) of the same concentration was given to the control group. ...
Objective: Connexin 43 (Cx43) is a protein constituent of gap junctions (GJs) in various barrier cells, especially astrocytes and microglia of the blood-brain-barrier (BBB), where it plays an important role in intercellular communication and regulation of the barrier. Despite the importance of Cx43 in other blood barriers, not much attention has been paid to expression and function of Cx43 in the blood-labyrinth-barrier (BLB) of the stria vascularis in the cochlea.
Methods: We used multiple research approaches, including immunocytochemical staining, patch-clamp dye loading technique, real-time quantitative reverse transcription (RT)-PCR, western blot, measurement of endocochlear potential (EP) with an electrode through the scala media, and auditory brainstem response to test hearing function.
Results: We found Cx43 expressed in vascular endothelial cells (ECs) and perivascular resident macrophages (PVMs) in the stria vascularis of adult C57BL/6 mouse cochleae. In particular, we found Cx43 expressed in foot processes of PVMs at points of contact with the endothelium. Consistent with Cx43 expression in vivo, we also found Cx43 expressed in EC-EC and EC-PVM interfaces in a co-cultured cell line model. Using a patch-clamp dye loading technique, we demonstrated that Alexa Fluor® 568 dye injected into PVMs diffuses to connected neighboring ECs. The functional coupling between the ECs and PVMs is blocked by 18α-Glycyrrhetinic acid (18α-GA), a GJ blocker. Suppression of Cx43 with small interfering RNA (siRNA) in vivo significantly elevated hearing threshold and caused the EP to drop and the blood barrier to become more permeable. In further study, using in vitro primary EC cell line models, we demonstrated that suppression of Cx43 disrupts intercellular tight junctions (TJs) in the EC monolayer and increases endothelial monolayer permeability.
Conculsion: Taken together, these findings underscore the importance of Cx43 expression in the normal ear for maintaining BLB integrity, normal EP, and hearing function.
... Other examples of non-viral delivery formulations, such as dendrimers, polyamine non-liposomal transfection reagents, and cell-penetrating peptides, have also been tested for inner ear gene delivery, but have not been used for therapy to date [21,96]. ...
... The authors showed efficient siRNA transfection to the cochlea of the chinchilla with this delivery system. They were able to demonstrate successful transfection of Cy3labeled siRNA into cells of the inner ear through the intact RWM, including the IHCs, OHCs, and vestibular cells in the crista ampullaris, macula utriculi, and macula sacculi (Qi et al., 2014). ...
Systemic delivery of therapeutics for targeting the cochlea to prevent or treat hearing loss is challenging. Systemic drugs have to cross the blood-labyrinth barrier (BLB). BLB can significantly prevent effective penetration of drugs in appropriate concentrations to protect against hearing loss caused by inflammation, ototoxic drugs, or acoustic trauma. This obstacle may be obviated by local administration of protective agents. This route can deliver higher concentration of drug compared to systemic application and preclude systemic side effects. Protective agents have been administered by intra-tympanic injection in numerous preclinical studies. Drugs such as steroids, etanercept, D and L-methionine, pifithrin-alpha, adenosine agonists, melatonin, kenpaullone (a cyclin-dependent kinase 2 (CDK2) inhibitor) have been reported to show efficacy against cisplatin ototoxicity in animal models. Several siRNAs have been shown to ameliorate cisplatin ototoxicity when administered by intra-tympanic injection. The application of corticosteroids and a number of other drugs with adjuvants appears to enhance efficacy. Administration of siRNAs to knock down AMPK kinase, liver kinase B1 (LKB1) or G9a in the cochlea have been found to ameliorate noise-induced hearing loss. The local administration of these compounds appears to be effective in protecting the cochlea against damage from cisplatin or noise trauma. Furthermore the intra-tympanic route yields maximum protection in the basal turn of the cochlea which is most vulnerable to cisplatin ototoxicity and noise trauma. There appears to be very little transfer of these agents to the systemic circulation. This would avoid potential side effects including interference with anti-tumor efficacy of cisplatin. Nanotechnology offers strategies to effectively deliver protective agents to the cochlea. This review summarizes the pharmacology of local drug delivery by intra-tympanic injection to prevent hearing loss caused by cisplatin and noise exposure in animals. Future refinements in local protective agents provide exciting prospects for amelioration of hearing loss resulting from cisplatin or noise exposure.
... Several studies have reported protection against ototoxic insults in vitro and ex vivo using RNAi [26][27][28][29]. Despite the successful delivery of siRNA to the cochlea [30,31], not many studies were successful in translating the outcome to in vivo studies. The areas of research using viral vector gene therapy or RNAi can be divided into genetic hearing loss, hearing protection, and HC regeneration. ...
Sensorineural hearing loss is caused by the loss of sensory hair cells (HCs) or a damaged afferent nerve pathway to the auditory cortex. The most common option for the treatment of sensorineural hearing loss is hearing rehabilitation using hearing devices. Various kinds of hearing devices are available but, despite recent advancements, their perceived sound quality does not mimic that of the “naïve” cochlea. Damage to crucial cochlear structures is mostly irreversible and results in permanent hearing loss. Cochlear HC regeneration has long been an important goal in the field of hearing research. However, it remains challenging because, thus far, no medical treatment has successfully regenerated cochlear HCs. Recent advances in genetic modulation and developmental techniques have led to novel approaches to generating HCs or protecting against HC loss, to preserve hearing. In this review, we present and review the current status of two different approaches to restoring or protecting hearing, gene therapy, including the newly introduced CRISPR/Cas9 genome editing, and stem cell therapy, and suggest the future direction.
... Because the mammalian inner ear is encased in the temporal bone, direct surgical intervention to access the membranous labyrinth is not trivial and can lead to unwanted side effects [3][4][5] . Established approaches to the perilymphatic or endolymphatic compartments include: (a) the perilymphatic approach, via a trans-round window membrane (RWM) injection [6][7][8] , cochleostomy to the scala tympani 9, 10 , (b) the endolymphatic approach, with a direct cochleostomy to the scala media 11, 12 , (c) or the semicircular canal canalostomy, which is likely a combination of the two pathways [13][14][15] (Fig. 1a). While the perilymphatic approach is relatively safe and commonly used for cochlear implantation in humans 16 , the endolymphatic approach is complex and carries a high risk of inner ear damage making it clinically unfeasible, although efforts are on-going to establish an atraumatic approach to the endolymphatic space in neonatal and adult murine models 12,17 . ...
Gene therapy for genetic deafness is a promising approach by which to prevent hearing loss or to restore hearing after loss has occurred. Although a variety of direct approaches to introduce viral particles into the inner ear have been described, presumed physiological barriers have heretofore precluded investigation of systemic gene delivery to the cochlea. In this study, we sought to characterize systemic delivery of a rAAV2/9 vector as a non-invasive means of cochlear transduction. In wild-type neonatal mice (postnatal day 0-1), we show that intravenous injection of rAAV2/9 carrying an eGFP-reporter gene results in binaural transduction of inner hair cells, spiral ganglion neurons and vestibular hair cells. Transduction efficiency increases in a dose-dependent manner. Inner hair cells are transduced in an apex-to-base gradient, with transduction reaching 96% in the apical turn. Hearing acuity in treated animals is unaltered at postnatal day 30. Transduction is influenced by viral serotype and age at injection, with less efficient cochlear transduction observed with systemic delivery of rAAV2/1 and in juvenile mice with rAAV2/9. Collectively, these data validate intravenous delivery of rAAV2/9 as a novel and atraumatic technique for inner ear transgene delivery in early postnatal mice.
... Inner ear hair cells (HCs), primary transducers for perception of sound and balance, are not regenerated in mammals once they are lost (Hawkins et al., 1976;Schacht, 1986), thus replacement using various medical strategies, such as gene or cell therapy, is required to improve hearing ability (de Felipe et al., 2011;Qi et al., 2014;Hu and Ulfendahl, 2013;Okano and Kelley, 2012). Forced expression of the transcription factor Math1 (also known as Atoh1) via viral infection has been reported to generate new HCs in vivo (Kawamoto et al., 2003;Zheng and Gao, 2000;Staecker et al., 2014;Husseman and Raphael, 2009), while induction of HC-like cells from various stem cell sources has also been achieved using novel methods (McLean et al., 2016;Elbana et al., 2015;Hartman et al., 2015;Bramhall et al., 2014) and applied as translational therapy for individuals with hearing loss (Li et al., 2004;Oiticica et al., 2010;Xu et al., 2016;Barboza et al., 2016;Jongkamonwiwat et al., 2010). ...
We sought to establish a more efficient technique for induction of inner ear hair cell-like cells (HC-like cells) from embryonic stem cells (ES cells) by using a combination of two previously reported methods; ST2 stromal cell-conditioned medium, known to be favorable for HC-like cell induction (HIST2 method), and ES cells with transfer of the Math1 gene (Math1-ES cells). Math1-ES cells carrying Tet-inducible Math1 were cultured for 14days with doxycycline in conditioned medium from cultures of ST2 stromal cells following formation of 4-day embryoid bodies (EBs). Although each of the previously introduced methods have been reported to induce approximately 20% HC-like cells and 10% HC-like cells in their respective populations in EB outgrowths at the end of the culture periods, the present combined method was able to generate approximately 30% HC-like cells expressing HC-related markers (myosin6, myosin7a, calretinin, α9AchR, Brn3c), which showed remarkable formation of stereocilia-like structures. Analysis of expressions of marker genes specific for cochlear (Lmod3, Emcn) and vestibular (Dnah5, Ptgds) cells indicated that our HIST2 method may lead to induction of cochlear- and vestibular-type cells. In addition, continuous Math1 induction by doxycycline without use of the HIST2 method preferentially induced cochlear markers with negligible effects on vestibular marker induction.
