[Breakthrough for deaf born children: First successful gene therapy for hearing impairment]

 Ellen Reisinger <sup>1

1</sup> Gene Therapy for Hearing Impairment, Department of Otorhinolaryngology Tübingen, and Gene and RNA Therapy Center (GRTC), Faculty of Medicine, University of Tübingen, Germany

Abstract

In several clinical studies to date (as of June 2024), more than a dozen children born deaf have been treated with a gene therapy developed specifically for this purpose – and can now hear with their own ears, dance to music, repeat words and answer questions. To achieve this, a gene supplementation therapy was used for children with OTOF-related deafness. This review article explains how well the children hear according to current knowledge and which hearing tests could provide further insights in follow-up studies. Finally, an outlook is given on the broader application of this gene therapy and the gene therapies under development for other forms of deafness.

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Design of the gene therapy for the cure of hearing impairment due to OTOF mutations

The successful gene therapy for OTOF-related deafness (DFNB9) marks the beginning of a new era: for the first time, it is possible to restore hearing to children who were previously effectively deaf. Virtually overnight, a congenital, genetic sensory impairment can now be treated causally, albeit currently only in clinical trials. In this form of deafness, synaptic transmission from the inner hair cells to the auditory nerve is impaired, which typically manifests itself in severe to profound hearing impairment with intact function of the outer hair cells, hence also called “auditory synaptopathy” [1], [2]. Therapeutically, the absence of the protein otoferlin, which is encoded by the gene OTOF, is compensated for by a gene supplementation therapy. Two adeno-associated viruses (AAVs) each introduce one half of the coding sequence for otoferlin into the inner hair cells. This “dual-AAV” or “split-AAV” strategy was necessary because the coding sequence was too long to be transported with one AAV [3], [4]. In the nucleus, the two coding sequences are assembled and ensure transcription and translation of the protein. First, independent studies had shown that when these therapeutic AAVs were injected into the inner ear of Otof-knockout mice, hearing was restored ([5], [6], later also in [7], [8]). In recent months, it has been reported at conferences and in scientific journals that this strategy also works in children born deaf: a few weeks after gene therapy hearing thresholds were at best 38 dB HL (pure tone average, PTA), and almost all of the children responded to their names and could repeat at least simple words [9], [10], [11].

How good is the hearing of children treated with the gene therapy?

Now that the first results of these clinical studies have been published, we should take a close look and use them wisely to further improve the gene therapy if necessary. The thresholds from auditory brainstem response recordings (ABR) and auditory steady state responses (ASSR) and, in older children, tone audiometry indicated hearing thresholds of 38–75 dB HL. However, only few studies present the raw ABR data, and even these are not compared with the ABR waves of children with normal hearing. For really good hearing results, however, it is crucial whether sufficient otoferlin protein is produced in the inner hair cells, which we cannot determine directly in the children. However, we know from the descriptions of affected individuals with (presumably) lower levels of otoferlin caused by point mutations in otoferlin which additional parameters in hearing assessments should be considered. For example, a two siblings with such a mutation, OTOF-p.Ile515Thr, showed only slightly increased hearing thresholds in tone audiometry, but clearly abnormal brainstem audiometry [12]. In the ABR, only wave V was recognizable, which was delayed and reduced in amplitude [13]. In a train of click stimuli, the first click was quite reliably represented in wave V, but the subsequent clicks were decreased in amplitude, so that wave V was less and less detectable in later clicks [13]. Adaptation to continuous stimuli was also strongly pronounced, more so in the high-frequency range than in the low-frequency range: these patients could barely perceive a (clearly suprathreshold) 8 kHz tone at the end of a 90 second auditory stimulus [13]. While speech comprehension for simple words reached 88–100%, it was severely impaired in everyday life and was less than 10% in background noise (HINT-C test) [12], [14]. In addition to the described siblings, similar hearing problems were found in five patients with other mutations in OTOF [15]. The underlying cellular and molecular dysfunction of this kind of hearing disorder was examined in more detail in a mouse model into which the Otof-p.Ile515Thr mutation was genetically introduced [16]. While a clear hearing impairment was found in brainstem audiometry, with reduced amplitudes and increased thresholds, the mice showed almost normal hearing thresholds in behavioral experiments [16]. Thus, the mouse model seems to recapitulate the phenotype of affected individuals well. Using immunohistochemistry, we found that the amount of otoferlin protein in the inner hair cells was reduced by about 65% [16]. Since otoferlin is required for synaptic transmission from hair cells to the auditory nerve [1], we also determined the extent to which synaptic function is impaired by the reduced amount of (mutated) otoferlin in this mouse line. It was found that the synapse was still able to respond appropriately to short stimuli, but the replenishment of synaptic vesicles was greatly slowed down during sustained stimuli [16]. As a result, at this synapse, a subsequent stimulus is only transmitted with the same intensity if the synapse can recover long enough. Applied to speech comprehension in background noise, this implies that the synaptic transmission at such a synapse is already exhausted by the background noise, so that additional signals from the speech can no longer be passed on. In addition, the synapses of a cell transmit a signal to the subsequent nerve cells with a wider temporal spread. This means that consonants are perceived as “blurred”, which can limit speech comprehension, even without background noise. Together, these findings explain why these and other affected people with a similar phenotype do not benefit from hearing aids: higher sound pressures do not improve speech comprehension because they lead to faster fatigue of the hair cell synapses [15].

We currently do not know how high the otoferlin levels are in the inner hair cells of children treated with gene therapy. Even if a deficit in the particularly sensitive hearing assessments would only indirectly indicate the amount of otoferlin and can also be caused by many factors, such tests should still be carried out in order to be able to work on improving the therapy itself or developing specific aftercare, such as targeted hearing training. In mice, otoferlin protein levels of approximately 35% of normal were measured after such gene therapy, which corresponds almost exactly to the amount of protein found in the Otof-p.Ile515Thr mice [4], [5]. In humans, this amount of protein could be higher or lower after gene therapy. In addition to potential problems with speech comprehension in noise, a slightly weaker performance of the inner hair cell synapse could also mean that those treated with gene therapy have to exert greater cognitive effort to decode the blurred speech signal. Due to the problem of a synapse that may exhaust more quickly, it is also unlikely that gene therapy-treated individuals benefit from hearing aids in order to correct the hearing thresholds of 38–75 dB HL, which are far from optimal – unless hearing aids are required to compensate for impaired cochlear amplification, which can be assessed by recording otoacoustic emissions (OAEs).

Which children with DFNB9 are eligible for this gene therapy?

Follow-up studies with more detailed measurements of hearing and speech comprehension will therefore have to show how good the gene therapy for OTOF deafness really is. In addition, the long-term observation of those children who have already been treated will be informative. The inclusion of more children will reveal any rare side effects and limitations that may occur. It is of great interest to many families of those affected whether gene therapy is also possible if a cochlear implant (CI) has already been inserted. The current gentle surgical procedures should in principle be suitable for ensuring that the sensory epithelium remains intact, which can be confirmed by measuring OAEs. If this is the case, gene therapy should be possible. A critical predictor of successful gene therapy for OTOF-induced deafness is therefore the presence of OAEs in both implanted and non-implanted ears. Even in non-implanted ears, OAEs are often lost within the first two years of life in OTOF patients; only in rare cases are OAEs detectable in adulthood [17], [18], [19]. In mouse models, it has also been shown that inner hair cells die in the absence of otoferlin; however, this has so far only been demonstrated in animal models [20]. The current clinical studies made the preserved OAEs an inclusion criterion and excluded CI-treated ears, as there is a risk that the gene therapy may not work as well as in non-implanted ears due to the implanted electrode and the resulting change in inner ear mechanics.

A plausible limitation for future studies and applications of gene therapy can be derived from the study by Lv et al. [9]: here, no improvement in hearing ability was observed in one of the 6 children treated. In contrast to the other children, this child had a pre-existing, albeit not very strong, immunity to the surface proteins of the viral vectors used here, the AAVs of serotype 1 (AAV1). In all children, immunity to the respective virus rose sharply within a few days of treatment [9], [10], [11]. The decisive factor for the success of gene therapy appears to be that the viral vectors reach their target cells before they are intercepted by the immune system. Once the viruses are in the cytoplasm, they are protected from direct attack by the cellular and humoral immune response. The surface proteins of the viruses are degraded and cannot be regenerated by these gene therapy viruses. The viral DNA, which consists mainly of the therapeutic gene, remains in the cell nucleus for many years. In non-dividing cells, which is the case for the sensory cells of the inner ear – it can therefore be assumed that once transduced, the cells will express the therapeutic gene for years, so that in the ideal case it is not necessary to repeat the treatment. However, the immunity awakened after a single injection of therapeutic viruses into the inner ear has significant implications for treatment planning: if only one ear is to be treated initially, the subsequent immunity to the surface proteins of the virus could prevent the second ear from being treated successfully – unless virus surface proteins from other serotypes that do not exhibit cross-immunity are used. Interestingly, at least two different variants of surface proteins are currently used in clinical trials, but the antibodies raised against those are at least partially cross-reactive [21]. The development of new serotypes that escape existing immunity is underway. With these it would be possible to treat the second ear using other gene therapy vectors. It would also be conceivable to inject another variant of gene therapy vectors into an already treated ear in order to increase the amount of otoferlin protein in the inner hair cells. Some overexpression by otoferlin-dual-AAV transduction of normal hearing mice and primates proved to be uncritical [5], [8], whereas an insufficient amount of protein presumably leads to the auditory fatigue described above.

Consequences of the new gene therapy for the diagnosis of hearing loss in newborns and infants

Despite possible limitations, this first successful gene therapy for deafness is a breakthrough. This will change how we diagnose and treat of children born deaf: while it was previously largely irrelevant which genetic disposition was the cause of the hearing loss, from now on a genetic analysis should be carried out – at least for those affected with auditory synaptopathy/neuropathy – in order to check whether gene therapy is available for this child as an alternative to cochlear implantation. However, since this form of deafness is overlooked in newborn hearing screenings based on OAEs [22], [23], it is now high time to re-evaluate the use of (more elaborate) auditory brainstem recordings in hearing screenings. In Germany, an estimated 60–90 children per year are born with auditory synaptopathy/neuropathy that can only be reliably detected with these procedures – including the approximately 15–25 children per year born with OTOF-related hearing loss [22], [23], [24], [25].

These studies are a breakthrough not only with regard to restoring hearing in these children: for the first time, the dual-AAV strategy has been used in humans, which allows large genes to be transported into cells with the help of small, non-pathogenic AAV viruses. Here, two pieces of DNA are injected into the ears with two different viruses, which reassemble in the nucleus of the cells and lead to transcription of the intact mRNA [26], [27], [28], [29]. OTOF gene therapy thus opens up new possibilities for other genetic diseases caused by large genes, not just limited to auditory disorders.

Which forms of hearing impairment will be treated with gene therapy next?

It would be highly desirable to be able to treat the most common genetic form of hearing impairment: the recessive form DFNB1, which is caused by mutations in the GJB2 gene and affects around 25% of children born deaf or hard of hearing [30]. This gene codes for the protein connexin26, which is required for the transport of potassium ions within the cochlea. However, there is a problem for the development of a gene therapy: for mice, the complete absence of the GJB2 gene is embryonically lethal [31]. Studies on newer mouse models, in which the GJB2 gene was inactivated only locally in the inner ear or only after the critical phase of embryonic development, indicate that connexin26 is required both during inner ear development and for the function of the mature inner ear [32]. However, it is not known why the same mutations lead to congenital severe to profound hearing loss in some affected individuals, but only mild to moderate, sometimes progressive hearing loss in approximately 25% of affected individuals [33]. Nevertheless, there is hope: the development of the new mouse models and a non-human primate model for GJB2 hearing loss by a company (Sensorion, Montpellier, France) will facilitate further research and enable gene therapy vectors to be tested. One difficulty for the development of this gene therapy will be that the gene should not be expressed in the sensory cells of the inner ear, which are particularly efficient at absorbing gene therapy viruses due to their high plasma membrane turnover. In this case, special virus constructs must be developed to achieve specific expression in the supporting cells and the stria vascularis. This is possible, but requires careful toxicological tests in non-human primate models, so that clinical trials are not imminent. The latter also applies to other forms of genetic hearing loss: while in many cases the affected genes are required during the development of the inner ear and would therefore have to be administered prenatally in humans, preclinical gene therapy studies with the gene TMPRSS3, which is affected in DFNB8/10, encountered the problem that overexpression proved to be toxic, and low expression were less effective [34]. It is therefore likely that a gene therapy for a second form of hearing loss that is successful in humans will be some time in coming.

Notes

Funding

I would like to thank the German Research Foundation (DFG) for support through the Heisenberg Program (project number 416097726).

Acknowledgments

I would like to thank Nicola Strenzke for her helpful comments on the manuscript during the review process.

Competing interests

The author claims to be co-author on a patent for dual-AAV gene therapy licensed by the University of Göttingen to Akouos Inc./Eli Lilly.

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