Overloaded Adeno-Associated Virus as a Novel Gene Therapeutic Tool for Otoferlin-Related Deafness

Abstract

Hearing impairment is the most common sensory disorder in humans. So far, rehabilitation of profoundly deaf subjects relies on direct stimulation of the auditory nerve through cochlear implants. However, in some forms of genetic hearing impairment, the organ of Corti is structurally intact and therapeutic replacement of the mutated gene could potentially restore near natural hearing. In the case of defects of the otoferlin gene (OTOF), such gene therapy is hindered by the size of the coding sequence (~6 kb) exceeding the cargo capacity (<5 kb) of the preferred viral vector, adeno-associated virus (AAV). Recently, a dual-AAV approach was used to partially restore hearing in deaf otoferlin knock-out (Otof-KO) mice. Here, we employed in vitro and in vivo approaches to assess the gene-therapeutic potential of naturally-occurring and newly-developed synthetic AAVs overloaded with the full-length Otof coding sequence. Upon early postnatal injection into the cochlea of Otof-KO mice, overloaded AAVs drove specific expression of otoferlin in ~30% of all IHCs, as demonstrated by immunofluorescence labeling and polymerase chain reaction. Recordings of auditory brainstem responses and a behavioral assay demonstrated partial restoration of hearing. Together, our results suggest that viral gene therapy of DFNB9—using a single overloaded AAV vector—is indeed feasible, reducing the complexity of gene transfer compared to dual-AAV approaches.

Cabp2-Gene Therapy Restores Inner Hair Cell Calcium Currents and Improves Hearing in a DFNB93 Mouse Model

“…To evaluate the impact of the chosen AAV capsids, we then performed the same experiments using the PHP.eB vector (titer: 8.2 × 10 12 genome copies/ml) that transduces hair cells with high efficiency ( Rankovic et al, 2021 ). Cochleae of injected animals were harvested at 5–6 weeks of age.…”

“…Mice of either sex were injected at postnatal day P5-7 using the round window approach as described in earlier studies ( Huet and Rankovic, 2021 ; Rankovic et al, 2021 ). In brief, anesthesia was obtained by isoflurane (5% for anesthesia induction, 2–3% for maintenance, and frequent testing of the absence of hind-limb withdrawal reflex) and analgesia using subdermal injection of buprenorphine (0.1 mg/kg body weight), and carprofen (5 mg/kg body weight, repeated 24 h after procedure) or application of Xylocain (10 mg spray).…”

“…Thus, novel methods for improved hearing restoration are currently being developed, including gene replacement, and editing or silencing as well as optogenetics (reviewed in Kleinlogel et al, 2020 ). In the recent years, the potential of a therapeutic approach using AAV-mediated gene transfer has been tested in a few animal deafness models (e.g., Akil et al, 2012 , 2019 ; Jung et al, 2015 ; Pan et al, 2017 ; Al-Moyed et al, 2019 ; Nist-Lund et al, 2019 ; Rankovic et al, 2021 ; Taiber et al, 2021 ).…”

Otoferlin Is Required for Proper Synapse Maturation and for Maintenance of Inner and Outer Hair Cells in Mouse Models for DFNB9

“…Recent experimental studies aim toward the development of a gene therapy to restore natural hearing in DFNB9 by transducing IHCs with the cDNA of otoferlin using adeno-associated viruses (AAVs). As the length of the otoferlin coding sequence exceeds the cargo capacity of AAVs, either an overload viral vector or a combination of two viruses (dual-AAV) were injected into the cochlea, which successfully rescued hearing function in Otof –/– mice ( Akil et al, 2019 ; Al-Moyed et al, 2019 ; Rankovic et al, 2020 ). Since overloading AAVs results in a mixture of truncated AAV genomes ( Dong et al, 2010 ; Lai et al, 2010 ; Wu et al, 2010 ), this technique is rather difficult to be translated to human application.…”

Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking

“…However, the large size and multiple domains of the protein have made establishing a mechanism for the protein challenging. The large size has also challenged traditional gene delivery strategies (Akil et al 2019;Cepeda et al 2018;Rankovic et al 2021). Recently several organismal model studies have suggested that the Cterminus of otoferlin is minimally necessary for function (Chatterjee et al 2015, Tertrais et al 2019.…”