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The process of hearing can be described as a series of events in which the energy of sound is transformed from vibrations in the air captured by structures of the outer ear (e.g. pinna) and further transferred through the ear canal to cause vibration of the tympanic membrane and ossicles of the middle ear, which then become traveling waves in the fluid contained within the cochlea. This leads to stimulation of the inner ear hair cells that, in turn, stimulate spiral ganglion neurons that transfer these signals to the primary auditory cortex, where these impulses are deciphered according to intensity and frequency. This complex combination of mechanosensory and physiological mechanisms involves many distinct types of cells, the function of which are impacted by numerous proteins, including those involved in ion channel activity, signal transduction and transcription. In the last 30 years, pathogenic variants in over 150 genes were found to be linked to hearing loss. Hearing loss affects over 460 million people world-wide, and current treatment approaches, such as hearing aids and cochlear implantations, serve to improve hearing capacity but do not address the underlying genetic cause of hearing loss. Therefore, therapeutic strategies designed to correct the genetic defects causative for hearing loss offer the possibility to treat these patients. In this review, we will discuss genetic causes of hearing loss, novel gene therapeutic strategies to correct hearing loss due to gene defects and some of the preclinical studies in hearing loss animal models as well as the clinical translation of gene therapy approaches to treat hearing loss patients.
Keywords: hearing loss, genetic variants, sequencing, gene therapy, genome editing
Introduction
Genetic diagnosis of hearing loss allows prognostic disease assessment and provides the opportunity for early therapeutic intervention. Identification of genes associated with hearing loss has led to deeper insight into the underlying biology of normal hearing and pathology of hearing loss. Coupled with our increased understanding of genetics and advances in molecular biology techniques, this information has greatly facilitated design of novel medical strategies to treat hereditary hearing loss, such as gene therapy approaches. Indeed, the first clinical trial testing gene therapy in hearing loss patients was initiated and there are several gene transfer modalities available for further evaluation and optimization.
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From genetic causes to novel therapeutics
Hearing is dependent upon the coordinated interaction of several cell types and their responses to mechanical and physiological stimuli. Especially cells of the sensory epithelium, including the inner and outer hair cells (HC) as well as spiral ganglion neurons (SGN) of the inner ear, play key roles in the hearing process (Figure 1). As with other human diseases, genetic aberrations that impact cellular functions can also lead to hearing loss.
Advances in sequencing technologies led to more rapid identification of genetic aberrations linked to hearing loss. Currently, pathogenic variants (PV) in more than 150 genes are known to be associated with hearing loss [42] (http://www.hereditaryhearingloss.org). Introduction of PV genotypes into various animal models has helped to reveal the biological mechanisms through which loss of functional proteins leads to hearing loss and has also led to improved understanding of normal hearing processes (Figure 1). For example, loss of function variants cause disruption of cellular and molecular processes necessary for normal hearing, such as inner ear sensory cell development, control of transmembrane potentials via ion channels, gap junction channels for maintenance of the endocochlear potential, glutamate signaling and alternative gene splicing events.
Identification of PV in hearing loss patients also allows the possibility for personalized medicine approaches such as gene therapy to treat these patients. Knowledge gained from previous experience using gene therapies to treat other human diseases can be exploited to adapt protocols for use in hearing loss patients. Here, some of the most relevant vector technologies available for gene therapy of the inner ear include adenoviral (AdV) vectors, adeno-associated viral (AAV) vectors and lentiviral vectors. Each of these vector systems has unique properties with potential advantages and disadvantages. Importantly, these vector systems are currently used to deliver advanced genome editing tools to correct PV in target cell populations and the relevance of this technology to repair genetic lesions in inner ear cells with the aim to improve hearing is a clinically relevant area to be exploited. In combination with the extensive information acquired from animal models, gene transfer and genomic modification technologies are expected to drive clinical development of novel gene therapy strategies to treat hearing loss patients.
Genetic alterations and clinical consequences
Hereditary hearing loss can be divided into two major subgroups, syndromic i.e. as a symptom of a superordinate disorder involving further anomalies of the ear and other organ systems and non-syndromic hearing loss (NSHL), involving only the function of the inner ear. Today more than 400 distinctive syndromes are well-defined, for which hearing loss is a characteristic mandatory or accessory symptom. Many non-distinctive syndromes and multi-systemic disorders comprise hearing loss as well. Approximately 70 % of genetically determined hearing loss is non-syndromic [2]. The by far most common genetic cause for hereditary NSHL are pathogenic variants affecting the gene GJB2, which account for roughly 10-30 % of NSHL [22, 46, 59], reaching over 50% in some ethnic groups [8]. Noteworthy, the majority of NSHL is distributed among more than 100 genes known to be associated with NSHL, and likely more will be identified. Hereditary hearing loss may follow an autosomal recessive (most frequently, about 80 % of NSHL), autosomal dominant, X-linked or mitochondrial inheritance pattern [2].
The underlying molecular changes are variable and comprise substantial numbers of truncating, missense and intronic variants as well as copy number variations (CNVs). While historically the genetic heterogeneity restricted testing to just a few genes, recent studies underline the benefits of comprehensive genetic testing by targeted sequencing panel analysis [45]. Against a background of steadily decreasing costs with continuously improving sequencing coverage as well as increasing reliability in computed CNV detection, it is only a question of time before whole exome sequencing (WES) and soon whole genome sequencing (WGS) will become the preferred method. There is no doubt that the interpretation of a large number of variants remains a major challenge in all of these techniques, as well as the task to handle and appropriately report a multitude of variants of unknown significance (VUS) [34]. The identification of the genetic cause becomes increasingly important regarding personalized prognosis and therapy selection [40]. Regarding the outcome of cochlear implant therapy, recipients with genetic alterations affecting the function of the cochlear sensory organ seem to perform significantly better in terms of speech recognition than patients with PV in genes associated with spiral ganglion neuron function [41].
Thus, all newborns and infants as well as children and young adults with confirmed (syndromic or non-syndromic) hearing loss without strong evidence for an environmental etiology should be seen by a geneticist and offered testing. In adults, a more detailed assessment of the development of hearing loss seems appropriate before recommending genetic counseling, although literature suggests that over 30% of adult onset progressive hearing loss that results in cochlear implantation has a genetic cause [33]. Once a causative PV is identified, genetic testing should be offered to family members at risk. Genetic counseling may also include assessment of recurrence risk for potential offspring [2]. While performing prenatal as well as preimplantation genetic diagnosis is generally feasible, this should be carefully considered with respect to the Deaf culture [42] and according to country-specific legal aspects. Due to the extreme heterogeneity of genetically determined hearing loss that accompanies the complex challenge of variant interpretation, general population-wide prenatal or newborn genetic screening is currently not recommended. However, in the future, screening for at least specific genetic alterations should be considered in addition to physiologic newborn screening [9, 43], with particular regards to targeted and potentially curative therapeutic approaches. Determination of genetic aberrations responsible for a patient´s hearing loss can be used to direct personalized approaches like gene therapy.
Vector options to introduce gene therapeutics into the inner ear
Generally, different concepts are available for gene therapy of hereditary hearing loss. To substitute for the function of a defective gene, an intact copy can be introduced into the relevant cells of the inner ear. Gene suppression, for example through expression of an shRNA that targets the transcript of the mutated gene to prevent its translation, can serve to eliminate dominant-negative effects that may interfere with proper cellular function even if an intact gene copy is provided. Finally, gene correction utilizing gene editing based on designer nuclease systems allows the specific removal of PV, thereby also keeping the natural regulation of gene expression via the physiologic promoter and chromatin environment.
Common to all different gene therapy strategies is the requirement for efficient transfer technologies to equip the target cells with expression units for the intact gene or miRNA, for shRNAs, or for the gene editing components. The complex 3D architecture and defined arrangement of the specific cell types inside the cochlea (see Figure 1) excludes ex-vivo cell manipulation and restricts treatment options to in vivo delivery systems. This is in contrast to other organ systems, such as the hematopoietic system, where stem cells can be extracted and re-infused into the patient upon ex-vivo genetic therapy. Viruses have evolutionarily co-evolved with their hosts and, as such, have developed specialized mechanisms to enter their target species and cell type(s). Therefore, viral vectors appear to be ideal vehicles to deliver genetic information to the cochlea. Furthermore, the different compartments in the cochlea are filled with lymph, which allows for the distribution of injected viral vector throughout the cochlea via this intracochlear fluid, while spread to other organs is theoretically limited to the enclosed organ system of the inner ear.
Several parameters are important for the success of viral-vector-based gene therapy approaches in the cochlea: (1) The vector volume that can be administered is limited. The outer wall of the inner ear is rigid, so that injection of too high vector volumes would increase the pressure and cause hydraulic trauma. Standard injection volumes are 1 µL in mice and are estimated to be 10-30 µL in humans. Thus, high-titer vector preparations are required to allow delivery in a small volume. One advantage for gene therapy application to the inner ear is that the total number of cells present in the cochlea is low as compared to other gene-therapy-relevant organ systems, so that a comparably low number of vector particles should suffice to achieve clinical benefit. (2) The endocochlear potential as a result of the different ion compositions of perilymph and endolymph is an important prerequisite for proper functioning of the hearing cascade. Thus, the buffer used to deliver vector preparations should be compatible with inner ear fluids and cell types. (3) Optimal delivery routes to administer viral vectors to the cochlea need to be investigated (Figure 2), and vector distribution and dissemination from the site of injection need to be characterized. (4) Pre-existing immunity to vector components, such as the capsid, or to transferred genes might limit gene transfer and/or expression efficiency, or cause local inflammation.
Currently, three main viral vector systems have emerged for inner ear gene therapy: (1) lentiviral (LV) vectors, (2) adenoviral vectors (AdV), and (3) adeno-associated viral (AAV) vectors. Each of these were tested in in vitro transduction experiments using cell lines, dissociated primary tissue and cochlear explants and were also characterized in vivo in rodent models. All three systems competently enter post-mitotic cells, and thus are suitable for inner ear cell transduction. Due to space limitations, we will primarily focus on LV and AAV vectors.
In contrast to AdV and AAV vector platforms, LV vectors stably anchor their genomic information into the host cells genome. While this feature is of great advantage when targeting dividing cells guaranteeing stable, long-term gene addition and transmission to daughter cells non-integrating vectors have a superior safety profile. Many of the specialized and treatment relevant otic cell types, such as hair cells (HC) and spiral ganglion neurons (SGN), are post-mitotic and thus compatible with non-integrating vector systems. Nevertheless, although naturally integration-competent, LV vectors can be rendered integration-deficient, e. g. upon catalytic inactivation of the viral integrase enzyme, creating so-called non-integrating LV (NILV) vectors. Although so far only the integrating LV vectors have been tested in the context of otic gene therapy settings, NILV vectors might emerge as attractive alternatives with improved safety profiles in the future.
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