Development and evaluation of helper dependent adenoviral vectors for inner ear gene delivery

Hearing loss impacts over 5% of the world population and has debilitating effects on communication which lead to increased rates of depression, anxiety, cognitive decline, and risk of dementia (Ahmed et al., 2017). Sensorineural hearing loss (SNHL) is the most common type of hearing loss that affects 466 million people worldwide (Olusanya et al., 2019). SNHL is due to loss or dysfunction of hair cells and spiral ganglion neurons, the two cell types essential for hearing (Ahmed et al., 2017). While our understanding of the genes and signaling cascades regulating the health and function of hair cells and spiral ganglion neurons continues to grow at an explosive pace (Dabdoub et al.), the ability to translate this knowledge into effective treatments for hearing loss lags far behind. To date, no FDA approved biological treatments exist to improve or restore auditory function. Hearing aids rehabilitate SNHL but do not restore cochlear function or stop hearing loss progression. Cochlear implants are the only way to restore hearing function in deaf patients, however they are limited in their ability to accurately transduce auditory information and are considered a last resort treatment (Mowry et al., 2012). Therefore, there is an urgent need to develop novel biological therapeutics to overcome the limited treatment strategies available to address this growing health crisis.

Due to recent clinical successes of viral vector gene therapy, it is a promising therapeutic approach to treat SNHL (Atkinson et al., 2014). In contrast to other therapeutic strategies, viral vectors have the potential to treat SNHL with a single administration by stably expressing a transgene(s) that corrects the underlying cause (Akil et al., 2012), protects against further deterioration, or enhances neural survival (Hashimoto et al., 2019). Currently, adeno-associated virus (AAV) is a leading candidate viral vector for inner ear gene therapy due to its low toxicity profile and ability to broadly transduce cells within the inner ear (Sacheli et al., 2013). Although AAV has tremendous potential for treating certain classes of SNHL, its small packaging capacity (∼4.8kb) restricts its use in treating many causes of SNHL. Although dual AAV therapy approaches will express transgenes that exceed the AAV packaging limit, these approaches can lead to highly variable results due to low efficiency and truncated transcripts that can result in expression of dominant negative mutants (McClements and MacLaren, 2017).

Recombinant Adenoviral vectors (Ad) have packaging capacities that are up to 7 times greater than AAV packaging capacities and have had great success in basic research and clinical applications. Furthermore, 1st generation Ad vectors can transduce a wide variety of inner ear cell types and clinical trial results (NCT02132130) have demonstrated their safety in the human inner ear. Although early generation Ad vectors are considered safe, they can lead to ototoxicity due to expression of proteins from the remaining viral genome (Kügler et al., 2003). Helper dependent adenoviral (HdAd) vectors are devoid of all viral genes and have a large packaging capacity (∼36 kb). HdAd vectors are safe, as preclinical trials with HdAd have demonstrated long-term correction in multiple mammalian disease models which include Duchenne muscular dystrophy, hyperbilirubinemia and Pompe's disease with a single administration of HdAd which was not possible with 1st generation Ad vectors (Rastall et al., 2016, Gilbert et al., 2003, Schmitt et al., 2014).

Despite the tremendous potential of HdAd, its use in the inner ear has been largely unexplored. Therefore, we set out to characterize HdAd transduction in the inner ear using delivery through the round window (RW) or scala media to assess differences in transduction seen with delivery to the perilymphatic and endolymphatic compartments. To do so we created an HdAd5 vector which infects cells through the Coxsackie-Adenovirus Receptor (CAR) and a chimeric HdAd 5/35 vector that uses the hCD46 receptor to infect cells. Subsequently, we analyzed HdAd5 transduction patterns in the inner ear of neonatal C57Bl6J and adult CBA/J mice. In parallel, we analyzed HdAd5/35 transduction in the inner ear of CBA/J and hCD46 transgenic mice to study the impact of hCD46 on inner ear transduction patterns. We found that RW delivery of HdAd5 lead to transduction in mesenchymal cells of the peri-lymphatic lining and modiolar region while scala media delivery leads to transduction within the organ of Corti. We found that RW delivery of HdAd 5/35 transduces cells in a similar pattern to HdAd5 but in a hCD46 dependent manner. Based on these data, we find that RW delivery of HdAd5 or HdAd5/35 does not lead to transduction of organ of Corti cells but scala media delivery of HdAd5 does. Taken together, our data indicate that HdAd5 and HdAd5/35 vectors are promising vectors for use in inner ear gene therapy to treat hearing loss. However, further developments that improve the ability of HdAd vectors to transduce the organ of Corti via RW delivery are needed.

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