Accepted Article Preview: Published ahead of advance online publication Potential treatments for genetic hearing loss in humans: current conundrums R Minoda, T Miwa, M Ise, H Takeda
Cite this article as: R Minoda, T Miwa, M Ise, H Takeda, Potential treatments for genetic hearing loss in humans: current conundrums, Gene Therapy accepted article preview 17 March 2015; doi: 10.1038/gt.2015.27. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Received 16 December 2014; revised 24 January 2015; accepted 12 February 2015; Accepted article preview online 17 March 2015
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Title: Potential treatments for genetic hearing loss in humans: current conundrums
Ryosei Minoda, Toru Miwa, Momoko Ise, Hiroki Takeda
Departments of Otolaryngology-Head and Neck Surgery, Kumamoto University, Graduate School of Medicine, 1-1-1 Honjo Chuouku, Kumamoto city, Kumamoto, Japan
Corresponding author: Ryosei Minoda, Ph.D., M.D. Department and institution: Department of Otolaryngology-Head and Neck Surgery, Kumamoto University, Graduate School of Medicine Correspondence should be addressed to R.M. ([email protected]). City and country: Kumamoto City, Japan Corresponding author’s address: 1-1-1 Honjo Chuoku Kumamoto, 860-0811, Japan Tel.: 81-96-373-5255 Fax: 81-96-373-5256 E-mail: [email protected]
Key Words, Genetic hearing loss, treatments, gene therapy, embryo therapy, neonatal therapy, ASO therapy Topic heading: Potential genetic hearing loss treatments
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Abstract Genetic defects are a major cause of hearing loss in newborns. Consequently, hearing loss has a profound negative impact on human daily living. Numerous causative genes for genetic hearing loss have been identified. However, presently, there are no truly curative treatments for this condition. There have been several recent reports on successful treatments in mice utilizing embryonic gene therapy, neonatal gene therapy and neonatal antisense oligonucleotide therapy. Herein, we describe state-of-the-art research on genetic hearing loss treatment through gene therapy and discuss the obstacles to overcome in curative treatments of genetic hearing loss in humans.
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Introduction Sensorineural hearing loss (SNHL) is the most common congenital disease in humans. The incidences of profound SNHL at birth in the United Kingdom and United States are 133 per 100,000 and 186 per 100,000 births, respectively.1 A genetic defect is the most common cause of hearing loss at birth and in childhood. More than half of all neonates with SNHL have inherited hearing loss. Approximately 70% of hereditary hearing loss cases are non-syndromic and approximately 30% are syndromic SNHL.2,3 Hereditary forms of genetic SNHL are as follows: 80% autosomal recessive, ~20% autosomal dominant, ~1% X-linked, and ≥1% via mitochondrial inheritance.4,5 Autosomal dominant SNHL often takes a post-lingual and progressive form, whereas autosomal recessive SNHL takes a pre-lingual form.5 The most common genetic cause of non-syndromic SNHL is a mutation within the GJB2 gene, which encodes connexin (Cx)26.6,7,8 The second most frequent cause of non-syndromic SNHL is a mutation in the GJB6 gene, which encodes Cx30.9,8 The most common form of syndromic SNHL is Pendred syndrome, which is associated with mutations within the solute carrier family 26 (SLC26A4) gene.10,11 Mutations in mitochondrial DNA can also induce syndromic and non-syndromic hearing loss.5,12 There are many reported causative genes of genetic hearing loss in addition to those described above.4 Although numerous causative genes for genetic hearing loss have been identified, there are no truly curative treatments for this condition yet. At present, treatments for SNHL only
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include hearing aids and/or cochlear implants. While hearing aids and cochlear implants are helpful treatments to compensate for hearing loss, they do not restore hearing to normal levels. There is still an urgent need for truly curative treatments. Recently, there have been several reports on successful treatment of genetic hearing loss caused by Cx30, vesicular glutamate transporter (VGLUT)3 and Usher syndrome 1c (USH1C) gene mutations, which are all known genetic causes of hearing loss in humans.4,13,14
1.
Normal inner ear anatomy and development
The inner ear has two basic functions: hearing, which occurs in the cochlea, and balancing, which occurs in the semi-circular canals and vestibule. The cochlea is divided into three compartments: the scala vestibule, scala tympani and scala media. The scala media, a part of the endolymphatic space, contains the organ of Corti (OC). The OC contains three cell populations: inner hair cells (IHCs), outer hair cells (OHCs) and supporting cells (SCs) (Fig. 1). Hair cells have stereocilia that emerge from their apical surface. Receptor potentials, generated by deflection of the stereocilia within the IHCs, induce neurotransmitter release at the synaptic ends.15 Therefore, sound waves are transmitted via the outer and middle ear to the inner ear fluid in the cochlea and transduced to electrical signals via IHCs. These signals are subsequently transmitted to the brain via efferent neurons and perceived as sound.
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The mammalian inner ear and its sensory neurons develop from the otic placode, a thickened patch of head ectoderm.16,17,18 Subsequently, one otocyst (per side) is formed by invagination of the otic placode at the level of the hindbrain at four weeks gestation in humans19 and embryonic day 9.5 (E9.5) in mice. Soon after, formation of the otocyst and neuroblasts delaminate from the ventral region of the otocyst. These neuroblasts will coalesce adjacent to the developing inner ear and begin to form the statoacoustic ganglion.20 By E12.5 in mice, the positions of the developing sensory patches, which form from a single common patch in the otocyst, can be identified.21,20,22 The cochlear part of the otocyst then begins to elongate into a spiral structure. The two and one-half turns of the coiled cochlea are not completed until 25 weeks gestation in humans,19 while the mouse cochlear duct has completed three-quarters of one turn around E13.5.
2.
A theorem of genetic hearing loss treatments
During development, appropriate spatiotemporal control of gene expression is necessary for normal development of the inner ear. During this process, expression of a gene begins; subsequently, the gene expression gradually becomes more widespread, and reaches a spatiotemporal maximum. Disruption of this process by a genetic mutation during inner ear development can cause genetic hearing loss. There are two major classes of genetic mutations:
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loss-of-function and gain-of-function. In loss-of-function mutations, the most common form, the protein product of a gene is either missing, non-functional, or reduced in level. These are typically recessive mutations, because a wild-type allele can usually compensate for the non-functional allele. In contrast, the altered gene product takes on a new molecular function in gain-of-function mutations, which usually follows dominant inheritance, because the presence of a normal allele is not capable of preventing the mutant allele from behaving abnormally. One gain-of-function mutation subtype is the dominant negative mutation, whereby the product of the mutant gene can compete with or inhibit the function of the wild-type product.23 There are three important time points to be cognizant of in genetic hearing loss treatments: (1) the target gene initiation point at which target gene expression begins in normal individuals (hereafter referred to as “normal gene initiation time”), (2) the time point at which the normal gene expression matures (hereafter referred to as “normal gene mature expression time”), and (3) the time point at which phenotypes begin to manifest after target gene deficiency (Fig. 2). Among treatments for genetic hearing loss caused by loss-of-function mutations, the most effective would be a gene redeeming treatment, which is matched precisely to the “normal gene initiation time.” However, before manifestation of the hearing loss phenotype, we might be able to treat genetic hearing loss by recovering reduced or missing functions via gene or protein transfer. Moreover, when the intention is to treat genetic hearing loss after phenotype
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manifestation, patients can likely be treated by redeeming genetic defects, in addition to regenerating damaged cochleae. Therefore, treatments initiated prior to hearing loss phenotype manifestation would be simpler and more effective. Data from neonatal hearing screening tests have demonstrated that the majority of SHNL patients can be detected utilizing this method.5 Indeed, hearing loss caused by Cx26 mutations, the most common genetic cause of non-syndromic SNHL, also usually presents with a congenital onset24. Therefore, embryonic treatments are inevitable if the treatment must be administered before hearing loss phenotype occurrence in such congenital genetic hearing loss patients. One important issue regarding embryonic treatments is the need to treat embryonic inner ears in the maternal uterus, which is technically feasible, but not facile. Thus, experiments targeting embryonic mouse inners ears are very complex and involved. Because of this intricateness, most animal studies on genetic hearing loss treatments have not targeted embryonic inner ears, but rather neonatal mouse inner ears. When we utilize rodents, particularly mice, to study genetic hearing loss treatments, we need to be aware of differences in development and maturation of auditory functions between rodents and humans. Auditory function initiates around postnatal day 13 (P13) in mice, but at 20 weeks gestation in humans.25,26 Even if an effective treatment is administered during the mouse neonatal period, the respective neonatal treatment would likely be ineffective in humans, since
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mouse neonatal treatment equates to human embryonic treatment. When considering treatment for genetic hearing loss caused by gain-of-function mutations, treatments in which new molecular functions are suppressed through RNA interference or degradation of the mutated gene product would likely be successful. As mentioned previously, timing is an important factor for determining the simplicity of a treatment and subsequent methods effective for loss-of-function mutations. This relationship in loss-of-function mutations is generally the same as those in gain-of-function mutations. One difference between loss-of-function mutations and gain-of-function mutations is that postnatal treatments may be more feasible in cases of genetic SNHL caused by a gain-of-function mutation, which typically occurs post-lingually;5 specifically, gain-of-function mutations usually do not present as hearing loss during the neonatal period. Additionally, neonatal hearing screening test data have demonstrated that approximately 15% of preschool children with SNHL show progressive hearing loss.27 In such genetic SNHL patients who do not present with hearing loss during the neonatal period, neonatal treatment may be more feasible for preventing subsequent hearing loss.
3.
Treatment for genetic hearing loss caused by a loss-of-function mutation
3-1. Embryonic treatments While it is an extremely intricate period of development, the embryonic stage is the ideal
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treatment period for genetic hearing loss. Miwa et al. reported successful treatments via transuterine gene transfer to the embryonic inner ear in Cx30 knockout mice.28 Cx proteins, which assemble to form vertebrate gap junctions, are crucial for auditory function.29 A large deletion within the Cx30 gene is the second most frequent cause of non-syndromic SNHL, as reported in nine countries.9,30 Homozygous Cx30-deletion mice have severe hearing impairment and demonstrate a complete loss of endocochlear potential (EP); EP represents the transepithelial difference in electric potential between the endolymphatic and perilymphatic compartments, and it is crucial for normal hearing function.31, 32 Miwa et al.28 aimed to determine whether embryonic gene transfer into the developing inner ear of Cx30-deficient mice could prevent manifestation of a subsequent hearing loss phenotype. They utilized electroporation-mediated transuterine gene transfer into otocysts (EUGO) at E11.5 and induced robust transgene expression in the cochleae of developing inner ears. Consequently, Miwa et al. showed that gene supplementation to insert the wild-type Cx30 gene into the otocysts of E11.5 Cx30-knockout mice prevented postnatal hearing loss (Fig. 3). Their results demonstrated that the induction of a target gene prior to the “normal gene initiation time” can prevent postnatal hearing loss. The Cx30 gene is first expressed around E14 in wild-type mice,33 and Cx30-deficient mice auditory functions begin to degenerate around P4.31 These findings suggest that transfection of wild-type Cx30 before the “normal gene initiation
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time” is effective at preventing subsequent hearing loss phenotype manifestation. These findings also suggest that precise timing of treatment with wild-type gene supplementation and “normal gene initiation time” may be unnecessary for achieving positive therapeutic effects.
3-2. Neonatal treatments Akil et al. reported that gene transfer of an adeno-associated virus serotype 1 (AAV1) vector at P1–P12 in the cochleae utilizing the wild-type VGLUT3 gene significantly ameliorated auditory function in VGLUT3-knockout mice.13 VGLUT3 deficiency has been shown to cause severe hearing loss by P10–P12 in mice due to the loss of glutamate release at IHC afferent synapses in the cochleae.34 In wild-type mice, VGLUT3 is not expressed in IHCs at E15, but is expressed by E19.34 Although there is no further detailed information about the timing of gene expression maturation, P1-P12 (the time at which Akil et al. performed gene transfer), corresponds to the timing after initial VGLUT3 gene expression in wild-type mice and probably also corresponds to the timing around or after the onset of the hearing loss phenotype manifestation. Akil et al. also reported that all mice that underwent gene transfer into the scala tympani via the round window membrane at P1-P3 exhibited a normal hearing threshold, and hearing levels were maintained in five out of 19 mice at nine months postnatal. Additionally, all mice that underwent gene transfer via the round window membrane at P10-P12 exhibited a normal hearing threshold as above, but
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hearing levels were maintained in only one out of 19 mice after 28 weeks postnatal. These findings suggest that AAV1-mediated VGLUT3 gene transfer at P1–P12 is effective at restoring hearing in VGULT3-deficient mice. Furthermore, earlier treatments are more effective than later treatments for maintaining ameliorated hearing levels. Generally, when considering genetic hearing loss treatments, one important issue to address is the determination of an appropriate strategy to induce gene transfection into the cochleae. Very few strategies are able to induce gene transfection effectively in the cochleae without any consequential damage.35 One feature of AAV vectors is that they are able to transfect IHCs via administration of AAV vectors into the scala tympani in the cochlea.36,37 Administration into the scala tympani is advantageous in that it is less traumatic compared with administration into the scala media, because the scala tympani does not contain sensory cells, while the scala media does (Fig. 1).38,36 Thus, AAV vectors could be the most efficient carrier as a treatment for hearing loss caused by VGLUT3 deficiency, since AAV vectors are able to effectively transfer VGLUT3 to IHCs less traumatically via scala tympanic administration. Recently, Yu et al. reported that hearing loss in conditional Cx26 knockout mice was untreatable by AAV vector-based neonatal gene therapy.39 They performed Cx26 gene transfer utilizing AAV serotype 2/1 hybrid vectors into the scala media of conditional Cx26 knockout mice at either P0 or P1, and found that Cx26 gene transfer via AAV vectors induced extensive
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Cx26 expression in cells lining the scala media of the cochleae. However, auditory brainstem responses did not show significant hearing amelioration. In wild-type mice, Cx26 is not detectable at E12 or E14 in the developing cochleae but is detectable by E16 in developing cochlea ducts, and by E18, this distribution is more widespread. 40 In conditional Cx26 knockout mice, degeneration of the cochlea is first detectable in Claudius cells around P8 and in OHCs around P13.41 Therefore, the neonatal treatment timing period adopted by Yu et al. may be after “normal gene mature expression time” but before phenotype manifestation. Thus, there were significant differences in the efficacy of AAV vector-based neonatal additive gene therapies between the reports by Akil et al. and Yu et al. that we will discuss in the next section.
4.
Treatment timing window (Fig. 2)
Choi et al.’s recent temporal expression study of doxycycline-inducible expression of Slc26a4 using transgenic mice revealed that forced expression of Slc26a4 from E0 to E16.5 completely prevented subsequent hearing loss, which diminished after E16.542 (Fig. 4). Slc26a4, which encodes the anion exchanger, pendrin, a transporter of anions such as Cl-, I- and HCO3-,43, 44 is a causative gene of Pendred syndrome, which involves development of thyroid goitres and SNHL. Normally, Slc26a4 protein (pendrin) expression begins in the cochlea at E16.5 at the basal turn of the cochlea and is detectable throughout the cochlea by E17.5.45 While Choi et al. did not intend to
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evaluate genetic hearing loss treatment per se, their results do provide us with valuable information on the appropriate treatment timing window for loss-of-function genetic hearing loss. Their data also suggest that the induction of normal target gene expression, before “normal gene initiation time”, can prevent putative postnatal hearing loss, and its treatment efficacy diminished rapidly after the “normal gene initiation time”. Their results further suggest that precise timing of treatment and normal gene initiation is probably unnecessary to achieve positive therapeutic effects. These results are consistent with data from Miwa,28 which demonstrated that otocystic gene transfer at E11.5 can prevent subsequent hearing loss in Cx30-deficient mice; normal Cx30 initial expression begins around E14. Choi et al. also reported that forcible expression of Slc26a4 after E18.5 was unable to prevent hearing loss (Fig. 4). Consequently, all mice showed severe to profound hearing loss.42 If we apply Choi’s finding to treatments of genetic hearing loss caused by loss-of function type mutations, their results suggest that gene redeeming therapy after “normal gene initiation time” appears to rapidly decrease its effectiveness. Thus, Choi et al.’s finding is almost consistent with Yu’s results in conditional Cx26 knockout mice, in which additive gene therapy after “normal gene mature expression time” was ineffective at hearing amelioration.41 However, Choi et al.’s findings are inconsistent with those of Akil et al. in VGLUT3-deficient mice, in which additive gene therapy around or even after the onset of hearing loss phenotype manifestation was effective
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for hearing amelioration.13 Although the exact reason for the discrepancy between Choi et al.’s and Akil et al.’s respective data remains elusive, there may be a significant difference regarding appropriate treatment timing windows depending on each specific gene. Each genetic hearing loss causative gene induces hearing loss via a different mechanism, and the treatable time window range for each gene might be affected by its respective mechanism. To summarize, precise timing of treatment and normal gene initiation is probably unnecessary to achieve positive therapeutic effects. Normal gene redeeming therapy before the “normal gene initiation time” is effective at preventing hearing loss caused by loss-of-function mutations, and its efficacy probably diminishes rapidly after the “normal gene initiation time”. Normal gene redeeming therapy after the “normal gene mature expression time” is probably ineffective. However, the efficacy of normal gene redeeming therapy after the “normal gene initiation time” and/or the “normal gene mature expression time” might vary depending on the causative genes.
5.
Locations that are crucial for genetic hearing loss treatment
As mentioned, during development, appropriate spatiotemporal control of gene expression is necessary for normal development of the inner ear. The pattern of gene expression largely varies by location. Ideally, precise matching of gene expression pattern by location would be preferable
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to achieve curative functional recovery without any harmful effects. There are several previous reports that provide valuable information regarding this issue. The Slc26a4 protein (pendrin) is first expressed in the normal murine inner ear at the following times: endolymphatic sac at E11.5; cochlea at 16.5 in the basal turn; saccule and utricle by 14.5; and ampullae by 16.5.45 Li et al.46 generated Slc26a4 transgenic mice, which expressed Slc26a4 only at the endolymphatic sac without detectable expression in the cochlea or vestibular organs. They showed that Slc26a4 expression in the endolymphatic sac successfully prevented anatomical abnormalities of the inner ear and postnatal auditory and vestibular dysfunction, which are commonly observed in Slc26a4-deleted mice. Their results also imply that pendrin supplementation at the endolymphatic sac is sufficient to treat Pendred syndrome in humans, which is caused by a Slc26a4 deficiency. Therapeutic gene transfers may cause gene expression in unintended areas, at locations lacking endogenous expression of the causative gene. Miwa et al. reported that EUGO induced a broader range of Cx30 gene expression than its original area of expression and caused no harmful effects on inner ear functions.28 Thus, it is likely unnecessary to match the ectopic location of a gene precisely with its endogenous area of expression. However, we do need to clarify the locations essential for curative treatments using gene transfer, and whether gene transfer in excessive areas causes harmful effects on inner ear functions for each causative gene.
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6.
Antisense oligonucleotide (ASO) treatment
Splicing mutations in the Ush1C gene, which encodes the protein harmonin, causes Usher syndrome type 1C, which involves congenital SNHL, vestibular dysfunction, and retinitis pigmentosa.14 The USH1C 216G>A mutation, a special cause of splicing mutations in Usher syndrome type 1C, creates an aberrant 5ʹ splice site in exon 3 of its pre-messenger RNA. Splicing mutations cause incorrect translation, thereby generating aberrant harmonin proteins and subsequently causing a loss-of-function type SNHL. Lentz et al. reported that SNHL in Ush1c216AA knock-in mice, which are useful animal models for Usher syndrome type 1C caused by the USH1C 216G>A mutation, is treatable using intraperitoneal administration of an ASO.14 ASOs are currently being tested in a number of clinical trials as treatments for muscular dystrophy, Crohn’s disease, and others.47,48 Lentz et al. selected the most effective ASO for correcting splicing mutations from several candidates via in vitro studies. ASOs were intraperitoneally injected into P3-P5 Ush1c216AA knock-in mice, and their auditory and vestibular functions were assessed at 1, 2 and 3 months of age. While control knock-in mice that were administered a scrambled ASO showed profound hearing loss and vestibular dysfunction, mice that received an effective ASO showed significant amelioration of auditory function at both low and mid frequencies, as well as of vestibular functions. Therefore, ASO treatment appears to be a very promising method for congenital hearing loss caused by splicing mutations. However,
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there are several issues that must be clarified and others that need to be resolved. First, we do not know the mechanism by which the intraperitoneally-injected ASOs are transmitted past both the blood-labyrinthine and blood-cochlea barriers into the inner ears. Second, we need to develop better curative treatments, since ASO treatment at P3-P5 demonstrated no amelioration at high frequencies.14 It has also been reported that harmonin, which is encoded by Ush1c, is detectable by E15 at the basal turn of the cochlea and is detectable at the apical turn as late as P30. 49 The absence of amelioration at high frequencies observed in Lentz et al.’s study may therefore be attributable to belated treatments following “normal harmonin initial gene expression” at the basal turn. In addition, to achieve better curative treatments utilizing ASOs, we should consider that the development and maturation of auditory functions in rodents occur later than do those in humans.25,26 Therefore, to achieve more curative treatments, the treatments should be performed at earlier stages, such as the embryonic stage.14
7.
Future directions
The above results regarding congenital genetic hearing loss suggest that normal gene redeeming treatments initiated prior to the “normal gene initiation time” are probably most effective at preventing subsequent hearing loss. Additionally, after the “normal gene initiation time”, the treatment efficacy probably diminishes significantly.42 However, the efficacy of normal gene
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redeeming therapy after the “normal gene initiation time” might vary depending on the causative genes.13 Considering the results of Lentz et al.,14 intraperitoneal administration of an embryonic ASO may be a simple and effective method to treat genetic SNHL caused by splicing mutations. However, wild-type gene supplementation during the embryonic stage is critical for curative treatment of the majority of genetic SNHL cases caused by loss-of-function mutations. To achieve this, we need to clarify and resolve several issues. First, there are limited methods available that can achieve gene transfer in embryonic inner ears at specific periods and locations. New transfer methods satisfying such conditions are necessary. Second, although access to the developing inner ear is necessary to achieve gene transfer, currently the only embryonic stage at which we can access developing mouse inner ears is E11.5 due to anatomical location. There are no reported methods that have accessed developing human inner ears successfully. Therefore, there is an urgent need to develop reliable and safe methods to manipulate developing inner ears at any stage, in both mice and humans. An ultrasound imaging system would likely be useful for this purpose (Fig. 5). Third, treatable time windows and essential locations of curative treatments for each gene have yet to be determined. Additionally, we have summarized the relationship between normal gene expression time in the cochlea and efficacy of normal gene redeeming therapy from the data from previous reports (Fig. 3). However, as mentioned above, Li et al.’s data46 suggest
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that Slc26a4 expression in the cochlea might not be essential for curative treatment. We need to clarify precisely the relationship between the locations essential for curative treatments and treatment timing. Fourth, to perform embryonic treatment, we must be able to precisely diagnose genetic hearing loss during the early embryonic stages. In humans, the otocysts are generated at five weeks gestation. Therefore, to perform embryonic treatment, precise information on the patient’s genetic mutations and functional outcomes caused by the genetic mutation must be obtained at the earliest gestational stage possible. Genetic testing using amniotic fluid, which currently is being performed clinically, would be highly useful for this purpose. In addition, it has been reported that foetal cell-free DNA can be isolated from maternal blood as early as the fifth week of gestation.50 Genetic testing using maternal blood could also be an attractive option for early genetic diagnosis and treatments in the future.51 There is a ray of hope for genetic hearing loss treatments, despite the many issues to overcome. Accumulating knowledge on the cellular and molecular mechanisms involved in inner ear function and morphology, in both mice and humans, will be important to develop clinically-relevant treatments.52 Although we did not discuss treatments utilizing stem cells, they are likely the best option for genetic SNHL treatment in patients following phenotypic manifestation.
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The authors have no conflict of interest directly relevant to the content of this article.
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Connexin30 (Gjb6)-deficiency causes severe hearing impairment and lack of endocochlear potential. Hum Mol Genet 2003; 12(1): 13-21. 32.
Michel V, Hardelin JP, Petit C. Molecular mechanism of a frequent genetic form of deafness. N Engl J Med 2003; 349(7): 716-7.
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Sun J, Ahmad S, Chen S, Tang W, Zhang Y, Chen P et al. Cochlear gap junctions coassembled from Cx26 and 30 show faster intercellular Ca2+ signaling than homomeric counterparts. Am J Physiol Cell Physiol 2005; 288(3): C613-23.
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Seal RP, Akil O, Yi E, Weber CM, Grant L, Yoo J et al. Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3. Neuron 2008; 57(2): 263-75.
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Fukui H, Raphael Y. Gene therapy for the inner ear. Hear Res 2013; 297: 99-105.
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Minoda R, Hayashida, M, Yumoto E. Gene transfer into the organ of Corti utilizing adeno-associated viral vectors via the scala tympani. Otol Jpn 200616: 397.
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Liu Y, Okada T, Sheykholeslami K, Shimazaki K, Nomoto T, Muramatsu S et al. Specific and efficient transduction of cochlear inner hair cells with recombinant adeno-associated virus type 3 vector. Mol Ther 2005; 12(4): 725-33.
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Minoda R, Yumoto E. Trans-uterine gene transfer into mouse otocysts. Toukeibu
Juritsushinkei 2006; 20: 33-37. 39.
Yu Q, Wang Y, Chang Q, Wang J, Gong S, Li H et al. Virally expressed connexin26 restores gap junction function in the cochlea of conditional Gjb2 knockout mice. Gene
Ther 2014; 21(1): 71-80. 40.
Frenz CM, Van De Water TR. Immunolocalization of connexin 26 in the developing mouse cochlea. Brain Res Brain Res Rev 2000; 32(1): 172-80.
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Cohen-Salmon M, Ott T, Michel V, Hardelin JP, Perfettini I, Eybalin M et al. Targeted ablation of connexin26 in the inner ear epithelial gap junction network causes hearing impairment and cell death. Curr Biol 2002; 12(13): 1106-11.
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42.
Choi BY, Kim HM, Ito T, Lee KY, Li X, Monahan K et al. Mouse model of enlarged vestibular aqueducts defines temporal requirement of Slc26a4 expression for hearing acquisition. J Clin Invest 2011; 121(11): 4516-25.
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Scott DA, Wang R, Kreman TM, Sheffield VC, Karniski LP. The Pendred syndrome gene encodes a chloride-iodide transport protein. Nat Genet 1999; 21(4): 440-3.
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Soleimani M, Greeley T, Petrovic S, Wang Z, Amlal H, Kopp P et al. Pendrin: an apical Cl-/OH-/HCO3- exchanger in the kidney cortex. Am J Physiol Renal Physiol 2001; 280(2): F356-64.
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Kim HM, Wangemann P. Epithelial cell stretching and luminal acidification lead to a retarded development of stria vascularis and deafness in mice lacking pendrin. PLoS
One 2011; 6(3): e17949. 46.
Li X, Sanneman JD, Harbidge DG, Zhou F, Ito T, Nelson R et al. SLC26A4 targeted to the endolymphatic sac rescues hearing and balance in Slc26a4 mutant mice. PLoS
Genet 2013; 9(7): e1003641. 47.
Miller TM, Pestronk A, David W, Rothstein J, Simpson E, Appel SH et al. An antisense oligonucleotide against SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral sclerosis: a phase 1, randomised, first-in-man study. Lancet Neurol 2013; 12(5): 435-42.
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Monteleone G, Fantini MC, Onali S, Zorzi F, Sancesario G, Bernardini S et al. Phase I clinical trial of Smad7 knockdown using antisense oligonucleotide in patients with active Crohn's disease. Mol Ther 2012; 20(4): 870-6.
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Boeda B, El-Amraoui A, Bahloul A, Goodyear R, Daviet L, Blanchard S et al. Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle. Embo J 2002; 21(24): 6689-99.
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Guibert J, Benachi A, Grebille AG, Ernault P, Zorn JR, Costa JM. Kinetics of SRY gene appearance in maternal serum: detection by real time PCR in early pregnancy after assisted reproductive technique. Hum Reprod 2003; 18(8): 1733-6.
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Lo YM. Non-invasive prenatal testing using massively parallel sequencing of maternal plasma DNA: from molecular karyotyping to fetal whole-genome sequencing. Reprod Biomed Online 2013; 27(6): 593-8.
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Minoda R, Izumikawa M, Kawamoto K, Raphael Y. Strategies for replacing lost cochlear hair cells. Neuroreport 2004; 15(7): 1089-92.
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Figure Legends Figure 1. Transverse section image of the cochlea. The adult mammalian cochlea was divided into three compartments: the scala vestibule, scala tympani and scala media. This image represents a cross-section of the scala media, which contains the organ of Corti (OC). The OC contains three cell populations: inner hair cells (IHCs), outer hair cells (OHCs) and supporting cells. The two types of auditory hair cells (IHCs and OHCs) play critical roles as mechano-electrical transducers for hearing. Auditory hair cells are covered by the tectorial membrane. The stria vascularis, located in the lateral wall of the scala media, is responsible for the secretion of K+ into the endolymph and for production of the endocochlear potential.
Figure 2. To treat genetic hearing loss caused by loss-of-function mutations, there are three important time points: (1) the target gene initiation point at which target gene expression begins in normal individuals (“normal gene initiation time”), (2) the time point at which the normal gene expression matures (“normal gene mature expression time”), and (3) the time point at which phenotypes begin to manifest after target gene deficiency. Miwa et al. demonstrated that otocystic gene transfer before the “normal gene initiation time” can prevent subsequent hearing loss in Cx30-deficient mice. Yu et al. reported that hearing loss in conditional Cx26-knockout mice was untreatable by AAV vector-based gene therapy in the
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neonatal period, which may be after the “normal gene mature expression time” but before phenotype manifestation. Akil et al. reported that AAV-mediated VGLUT3 gene transfer at P1–P12, which corresponds to the time after initial VGLUT3 gene expression in wild type mice and probably also corresponds to the time around or after onset of the hearing loss phenotype manifestation, is effective at restoring hearing in VGULT3-deficient mice. Choi et al. reported that forced expression of Slc26a4 before the “normal gene initiation time” completely prevented subsequent hearing loss, which was diminished after the “normal gene initiation time”. The solid blue lines with arrows indicate successful functional recovery treatment time windows; the solid orange line with arrows indicates a period during which treatment is partially effective. The dotted black lines with arrows indicate the periods during which treatments are ineffective.
Figure 3. The embryonic stage is the ideal period for genetic hearing loss treatment. Miwa et al. reported successful treatment via transuterine gene transfer into the embryonic inner ear in Cx30-knockout mice. Gene transfer was performed by embryonic gene transfer into the developing inner ear in Cx30-deficient mice, which successfully prevented subsequent hearing loss phenotypic manifestation. Electroporation-mediated transuterine gene transfer was performed in otocysts (EUGO) in Cx30-deficient mice at E11.5. Embryos were delivered via
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C-section at E18.5, and the pups that underwent gene transfer at E11.5 were passed to surrogate dams to raise said embryos; these pups did not demonstrate hearing loss at P30.
Figure 4. Choi et al. reported that temporal expression of doxycycline-inducible solute carrier family 26 (Slc26a4) at E0-E16.5 in transgenic mice prevented subsequent hearing loss. The line in the figure indicates the relationship between onset of forced Slc26a4 expression and the consequent hearing threshold at one month post-birth. The efficacy of forced expression for preventing putative postnatal hearing loss decreased rapidly after E16.5, at which Slc26a4 expression begins at the basal turn of the cochlea.
Figure 5. a) Sagittal section of E14.5 mouse embryos in the uterus, observed via a small animal ultrasound imaging system with spatial resolution up to 30 µm (Prospect, S-Shape Corporation, New Taipei, Taiwan). The outline of the embryo is clearly detectable. The asterisk indicates the mouth. Arrowheads indicate the uterine wall. The dotted line indicates the plane on which the embryo’s head was observed in Figure 5b. b) Horizontal section images of the head of an E14.5 mouse embryo in the uterus. The arrow indicates the eye. The asterisk indicates the auricle. The circled area represents the cochlea. The inset image in the right corner shows a magnified image of the cochlea; arrow heads indicate the cochlea. Thus, detection of the cochlea utilizing the
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ultrasound imaging system may be feasible, but the approach to the developing inner ear does not appear to be facile.
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Cite this article as: R Minoda, T Miwa, M Ise, H Takeda, Potential treatments for genetic hearing loss in humans: current conundrums, Gene Therapy accepted article preview 17 March 2015; doi: 10.1038/gt.2015.27. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Received 16 December 2014; revised 24 January 2015; accepted 12 February 2015; Accepted article preview online 17 March 2015
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Title: Potential treatments for genetic hearing loss in humans: current conundrums
Ryosei Minoda, Toru Miwa, Momoko Ise, Hiroki Takeda
Departments of Otolaryngology-Head and Neck Surgery, Kumamoto University, Graduate School of Medicine, 1-1-1 Honjo Chuouku, Kumamoto city, Kumamoto, Japan
Corresponding author: Ryosei Minoda, Ph.D., M.D. Department and institution: Department of Otolaryngology-Head and Neck Surgery, Kumamoto University, Graduate School of Medicine Correspondence should be addressed to R.M. ([email protected]). City and country: Kumamoto City, Japan Corresponding author’s address: 1-1-1 Honjo Chuoku Kumamoto, 860-0811, Japan Tel.: 81-96-373-5255 Fax: 81-96-373-5256 E-mail: [email protected]
Key Words, Genetic hearing loss, treatments, gene therapy, embryo therapy, neonatal therapy, ASO therapy Topic heading: Potential genetic hearing loss treatments
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Abstract Genetic defects are a major cause of hearing loss in newborns. Consequently, hearing loss has a profound negative impact on human daily living. Numerous causative genes for genetic hearing loss have been identified. However, presently, there are no truly curative treatments for this condition. There have been several recent reports on successful treatments in mice utilizing embryonic gene therapy, neonatal gene therapy and neonatal antisense oligonucleotide therapy. Herein, we describe state-of-the-art research on genetic hearing loss treatment through gene therapy and discuss the obstacles to overcome in curative treatments of genetic hearing loss in humans.
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Introduction Sensorineural hearing loss (SNHL) is the most common congenital disease in humans. The incidences of profound SNHL at birth in the United Kingdom and United States are 133 per 100,000 and 186 per 100,000 births, respectively.1 A genetic defect is the most common cause of hearing loss at birth and in childhood. More than half of all neonates with SNHL have inherited hearing loss. Approximately 70% of hereditary hearing loss cases are non-syndromic and approximately 30% are syndromic SNHL.2,3 Hereditary forms of genetic SNHL are as follows: 80% autosomal recessive, ~20% autosomal dominant, ~1% X-linked, and ≥1% via mitochondrial inheritance.4,5 Autosomal dominant SNHL often takes a post-lingual and progressive form, whereas autosomal recessive SNHL takes a pre-lingual form.5 The most common genetic cause of non-syndromic SNHL is a mutation within the GJB2 gene, which encodes connexin (Cx)26.6,7,8 The second most frequent cause of non-syndromic SNHL is a mutation in the GJB6 gene, which encodes Cx30.9,8 The most common form of syndromic SNHL is Pendred syndrome, which is associated with mutations within the solute carrier family 26 (SLC26A4) gene.10,11 Mutations in mitochondrial DNA can also induce syndromic and non-syndromic hearing loss.5,12 There are many reported causative genes of genetic hearing loss in addition to those described above.4 Although numerous causative genes for genetic hearing loss have been identified, there are no truly curative treatments for this condition yet. At present, treatments for SNHL only
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include hearing aids and/or cochlear implants. While hearing aids and cochlear implants are helpful treatments to compensate for hearing loss, they do not restore hearing to normal levels. There is still an urgent need for truly curative treatments. Recently, there have been several reports on successful treatment of genetic hearing loss caused by Cx30, vesicular glutamate transporter (VGLUT)3 and Usher syndrome 1c (USH1C) gene mutations, which are all known genetic causes of hearing loss in humans.4,13,14
1.
Normal inner ear anatomy and development
The inner ear has two basic functions: hearing, which occurs in the cochlea, and balancing, which occurs in the semi-circular canals and vestibule. The cochlea is divided into three compartments: the scala vestibule, scala tympani and scala media. The scala media, a part of the endolymphatic space, contains the organ of Corti (OC). The OC contains three cell populations: inner hair cells (IHCs), outer hair cells (OHCs) and supporting cells (SCs) (Fig. 1). Hair cells have stereocilia that emerge from their apical surface. Receptor potentials, generated by deflection of the stereocilia within the IHCs, induce neurotransmitter release at the synaptic ends.15 Therefore, sound waves are transmitted via the outer and middle ear to the inner ear fluid in the cochlea and transduced to electrical signals via IHCs. These signals are subsequently transmitted to the brain via efferent neurons and perceived as sound.
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The mammalian inner ear and its sensory neurons develop from the otic placode, a thickened patch of head ectoderm.16,17,18 Subsequently, one otocyst (per side) is formed by invagination of the otic placode at the level of the hindbrain at four weeks gestation in humans19 and embryonic day 9.5 (E9.5) in mice. Soon after, formation of the otocyst and neuroblasts delaminate from the ventral region of the otocyst. These neuroblasts will coalesce adjacent to the developing inner ear and begin to form the statoacoustic ganglion.20 By E12.5 in mice, the positions of the developing sensory patches, which form from a single common patch in the otocyst, can be identified.21,20,22 The cochlear part of the otocyst then begins to elongate into a spiral structure. The two and one-half turns of the coiled cochlea are not completed until 25 weeks gestation in humans,19 while the mouse cochlear duct has completed three-quarters of one turn around E13.5.
2.
A theorem of genetic hearing loss treatments
During development, appropriate spatiotemporal control of gene expression is necessary for normal development of the inner ear. During this process, expression of a gene begins; subsequently, the gene expression gradually becomes more widespread, and reaches a spatiotemporal maximum. Disruption of this process by a genetic mutation during inner ear development can cause genetic hearing loss. There are two major classes of genetic mutations:
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loss-of-function and gain-of-function. In loss-of-function mutations, the most common form, the protein product of a gene is either missing, non-functional, or reduced in level. These are typically recessive mutations, because a wild-type allele can usually compensate for the non-functional allele. In contrast, the altered gene product takes on a new molecular function in gain-of-function mutations, which usually follows dominant inheritance, because the presence of a normal allele is not capable of preventing the mutant allele from behaving abnormally. One gain-of-function mutation subtype is the dominant negative mutation, whereby the product of the mutant gene can compete with or inhibit the function of the wild-type product.23 There are three important time points to be cognizant of in genetic hearing loss treatments: (1) the target gene initiation point at which target gene expression begins in normal individuals (hereafter referred to as “normal gene initiation time”), (2) the time point at which the normal gene expression matures (hereafter referred to as “normal gene mature expression time”), and (3) the time point at which phenotypes begin to manifest after target gene deficiency (Fig. 2). Among treatments for genetic hearing loss caused by loss-of-function mutations, the most effective would be a gene redeeming treatment, which is matched precisely to the “normal gene initiation time.” However, before manifestation of the hearing loss phenotype, we might be able to treat genetic hearing loss by recovering reduced or missing functions via gene or protein transfer. Moreover, when the intention is to treat genetic hearing loss after phenotype
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manifestation, patients can likely be treated by redeeming genetic defects, in addition to regenerating damaged cochleae. Therefore, treatments initiated prior to hearing loss phenotype manifestation would be simpler and more effective. Data from neonatal hearing screening tests have demonstrated that the majority of SHNL patients can be detected utilizing this method.5 Indeed, hearing loss caused by Cx26 mutations, the most common genetic cause of non-syndromic SNHL, also usually presents with a congenital onset24. Therefore, embryonic treatments are inevitable if the treatment must be administered before hearing loss phenotype occurrence in such congenital genetic hearing loss patients. One important issue regarding embryonic treatments is the need to treat embryonic inner ears in the maternal uterus, which is technically feasible, but not facile. Thus, experiments targeting embryonic mouse inners ears are very complex and involved. Because of this intricateness, most animal studies on genetic hearing loss treatments have not targeted embryonic inner ears, but rather neonatal mouse inner ears. When we utilize rodents, particularly mice, to study genetic hearing loss treatments, we need to be aware of differences in development and maturation of auditory functions between rodents and humans. Auditory function initiates around postnatal day 13 (P13) in mice, but at 20 weeks gestation in humans.25,26 Even if an effective treatment is administered during the mouse neonatal period, the respective neonatal treatment would likely be ineffective in humans, since
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mouse neonatal treatment equates to human embryonic treatment. When considering treatment for genetic hearing loss caused by gain-of-function mutations, treatments in which new molecular functions are suppressed through RNA interference or degradation of the mutated gene product would likely be successful. As mentioned previously, timing is an important factor for determining the simplicity of a treatment and subsequent methods effective for loss-of-function mutations. This relationship in loss-of-function mutations is generally the same as those in gain-of-function mutations. One difference between loss-of-function mutations and gain-of-function mutations is that postnatal treatments may be more feasible in cases of genetic SNHL caused by a gain-of-function mutation, which typically occurs post-lingually;5 specifically, gain-of-function mutations usually do not present as hearing loss during the neonatal period. Additionally, neonatal hearing screening test data have demonstrated that approximately 15% of preschool children with SNHL show progressive hearing loss.27 In such genetic SNHL patients who do not present with hearing loss during the neonatal period, neonatal treatment may be more feasible for preventing subsequent hearing loss.
3.
Treatment for genetic hearing loss caused by a loss-of-function mutation
3-1. Embryonic treatments While it is an extremely intricate period of development, the embryonic stage is the ideal
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treatment period for genetic hearing loss. Miwa et al. reported successful treatments via transuterine gene transfer to the embryonic inner ear in Cx30 knockout mice.28 Cx proteins, which assemble to form vertebrate gap junctions, are crucial for auditory function.29 A large deletion within the Cx30 gene is the second most frequent cause of non-syndromic SNHL, as reported in nine countries.9,30 Homozygous Cx30-deletion mice have severe hearing impairment and demonstrate a complete loss of endocochlear potential (EP); EP represents the transepithelial difference in electric potential between the endolymphatic and perilymphatic compartments, and it is crucial for normal hearing function.31, 32 Miwa et al.28 aimed to determine whether embryonic gene transfer into the developing inner ear of Cx30-deficient mice could prevent manifestation of a subsequent hearing loss phenotype. They utilized electroporation-mediated transuterine gene transfer into otocysts (EUGO) at E11.5 and induced robust transgene expression in the cochleae of developing inner ears. Consequently, Miwa et al. showed that gene supplementation to insert the wild-type Cx30 gene into the otocysts of E11.5 Cx30-knockout mice prevented postnatal hearing loss (Fig. 3). Their results demonstrated that the induction of a target gene prior to the “normal gene initiation time” can prevent postnatal hearing loss. The Cx30 gene is first expressed around E14 in wild-type mice,33 and Cx30-deficient mice auditory functions begin to degenerate around P4.31 These findings suggest that transfection of wild-type Cx30 before the “normal gene initiation
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time” is effective at preventing subsequent hearing loss phenotype manifestation. These findings also suggest that precise timing of treatment with wild-type gene supplementation and “normal gene initiation time” may be unnecessary for achieving positive therapeutic effects.
3-2. Neonatal treatments Akil et al. reported that gene transfer of an adeno-associated virus serotype 1 (AAV1) vector at P1–P12 in the cochleae utilizing the wild-type VGLUT3 gene significantly ameliorated auditory function in VGLUT3-knockout mice.13 VGLUT3 deficiency has been shown to cause severe hearing loss by P10–P12 in mice due to the loss of glutamate release at IHC afferent synapses in the cochleae.34 In wild-type mice, VGLUT3 is not expressed in IHCs at E15, but is expressed by E19.34 Although there is no further detailed information about the timing of gene expression maturation, P1-P12 (the time at which Akil et al. performed gene transfer), corresponds to the timing after initial VGLUT3 gene expression in wild-type mice and probably also corresponds to the timing around or after the onset of the hearing loss phenotype manifestation. Akil et al. also reported that all mice that underwent gene transfer into the scala tympani via the round window membrane at P1-P3 exhibited a normal hearing threshold, and hearing levels were maintained in five out of 19 mice at nine months postnatal. Additionally, all mice that underwent gene transfer via the round window membrane at P10-P12 exhibited a normal hearing threshold as above, but
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hearing levels were maintained in only one out of 19 mice after 28 weeks postnatal. These findings suggest that AAV1-mediated VGLUT3 gene transfer at P1–P12 is effective at restoring hearing in VGULT3-deficient mice. Furthermore, earlier treatments are more effective than later treatments for maintaining ameliorated hearing levels. Generally, when considering genetic hearing loss treatments, one important issue to address is the determination of an appropriate strategy to induce gene transfection into the cochleae. Very few strategies are able to induce gene transfection effectively in the cochleae without any consequential damage.35 One feature of AAV vectors is that they are able to transfect IHCs via administration of AAV vectors into the scala tympani in the cochlea.36,37 Administration into the scala tympani is advantageous in that it is less traumatic compared with administration into the scala media, because the scala tympani does not contain sensory cells, while the scala media does (Fig. 1).38,36 Thus, AAV vectors could be the most efficient carrier as a treatment for hearing loss caused by VGLUT3 deficiency, since AAV vectors are able to effectively transfer VGLUT3 to IHCs less traumatically via scala tympanic administration. Recently, Yu et al. reported that hearing loss in conditional Cx26 knockout mice was untreatable by AAV vector-based neonatal gene therapy.39 They performed Cx26 gene transfer utilizing AAV serotype 2/1 hybrid vectors into the scala media of conditional Cx26 knockout mice at either P0 or P1, and found that Cx26 gene transfer via AAV vectors induced extensive
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Cx26 expression in cells lining the scala media of the cochleae. However, auditory brainstem responses did not show significant hearing amelioration. In wild-type mice, Cx26 is not detectable at E12 or E14 in the developing cochleae but is detectable by E16 in developing cochlea ducts, and by E18, this distribution is more widespread. 40 In conditional Cx26 knockout mice, degeneration of the cochlea is first detectable in Claudius cells around P8 and in OHCs around P13.41 Therefore, the neonatal treatment timing period adopted by Yu et al. may be after “normal gene mature expression time” but before phenotype manifestation. Thus, there were significant differences in the efficacy of AAV vector-based neonatal additive gene therapies between the reports by Akil et al. and Yu et al. that we will discuss in the next section.
4.
Treatment timing window (Fig. 2)
Choi et al.’s recent temporal expression study of doxycycline-inducible expression of Slc26a4 using transgenic mice revealed that forced expression of Slc26a4 from E0 to E16.5 completely prevented subsequent hearing loss, which diminished after E16.542 (Fig. 4). Slc26a4, which encodes the anion exchanger, pendrin, a transporter of anions such as Cl-, I- and HCO3-,43, 44 is a causative gene of Pendred syndrome, which involves development of thyroid goitres and SNHL. Normally, Slc26a4 protein (pendrin) expression begins in the cochlea at E16.5 at the basal turn of the cochlea and is detectable throughout the cochlea by E17.5.45 While Choi et al. did not intend to
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evaluate genetic hearing loss treatment per se, their results do provide us with valuable information on the appropriate treatment timing window for loss-of-function genetic hearing loss. Their data also suggest that the induction of normal target gene expression, before “normal gene initiation time”, can prevent putative postnatal hearing loss, and its treatment efficacy diminished rapidly after the “normal gene initiation time”. Their results further suggest that precise timing of treatment and normal gene initiation is probably unnecessary to achieve positive therapeutic effects. These results are consistent with data from Miwa,28 which demonstrated that otocystic gene transfer at E11.5 can prevent subsequent hearing loss in Cx30-deficient mice; normal Cx30 initial expression begins around E14. Choi et al. also reported that forcible expression of Slc26a4 after E18.5 was unable to prevent hearing loss (Fig. 4). Consequently, all mice showed severe to profound hearing loss.42 If we apply Choi’s finding to treatments of genetic hearing loss caused by loss-of function type mutations, their results suggest that gene redeeming therapy after “normal gene initiation time” appears to rapidly decrease its effectiveness. Thus, Choi et al.’s finding is almost consistent with Yu’s results in conditional Cx26 knockout mice, in which additive gene therapy after “normal gene mature expression time” was ineffective at hearing amelioration.41 However, Choi et al.’s findings are inconsistent with those of Akil et al. in VGLUT3-deficient mice, in which additive gene therapy around or even after the onset of hearing loss phenotype manifestation was effective
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for hearing amelioration.13 Although the exact reason for the discrepancy between Choi et al.’s and Akil et al.’s respective data remains elusive, there may be a significant difference regarding appropriate treatment timing windows depending on each specific gene. Each genetic hearing loss causative gene induces hearing loss via a different mechanism, and the treatable time window range for each gene might be affected by its respective mechanism. To summarize, precise timing of treatment and normal gene initiation is probably unnecessary to achieve positive therapeutic effects. Normal gene redeeming therapy before the “normal gene initiation time” is effective at preventing hearing loss caused by loss-of-function mutations, and its efficacy probably diminishes rapidly after the “normal gene initiation time”. Normal gene redeeming therapy after the “normal gene mature expression time” is probably ineffective. However, the efficacy of normal gene redeeming therapy after the “normal gene initiation time” and/or the “normal gene mature expression time” might vary depending on the causative genes.
5.
Locations that are crucial for genetic hearing loss treatment
As mentioned, during development, appropriate spatiotemporal control of gene expression is necessary for normal development of the inner ear. The pattern of gene expression largely varies by location. Ideally, precise matching of gene expression pattern by location would be preferable
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to achieve curative functional recovery without any harmful effects. There are several previous reports that provide valuable information regarding this issue. The Slc26a4 protein (pendrin) is first expressed in the normal murine inner ear at the following times: endolymphatic sac at E11.5; cochlea at 16.5 in the basal turn; saccule and utricle by 14.5; and ampullae by 16.5.45 Li et al.46 generated Slc26a4 transgenic mice, which expressed Slc26a4 only at the endolymphatic sac without detectable expression in the cochlea or vestibular organs. They showed that Slc26a4 expression in the endolymphatic sac successfully prevented anatomical abnormalities of the inner ear and postnatal auditory and vestibular dysfunction, which are commonly observed in Slc26a4-deleted mice. Their results also imply that pendrin supplementation at the endolymphatic sac is sufficient to treat Pendred syndrome in humans, which is caused by a Slc26a4 deficiency. Therapeutic gene transfers may cause gene expression in unintended areas, at locations lacking endogenous expression of the causative gene. Miwa et al. reported that EUGO induced a broader range of Cx30 gene expression than its original area of expression and caused no harmful effects on inner ear functions.28 Thus, it is likely unnecessary to match the ectopic location of a gene precisely with its endogenous area of expression. However, we do need to clarify the locations essential for curative treatments using gene transfer, and whether gene transfer in excessive areas causes harmful effects on inner ear functions for each causative gene.
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6.
Antisense oligonucleotide (ASO) treatment
Splicing mutations in the Ush1C gene, which encodes the protein harmonin, causes Usher syndrome type 1C, which involves congenital SNHL, vestibular dysfunction, and retinitis pigmentosa.14 The USH1C 216G>A mutation, a special cause of splicing mutations in Usher syndrome type 1C, creates an aberrant 5ʹ splice site in exon 3 of its pre-messenger RNA. Splicing mutations cause incorrect translation, thereby generating aberrant harmonin proteins and subsequently causing a loss-of-function type SNHL. Lentz et al. reported that SNHL in Ush1c216AA knock-in mice, which are useful animal models for Usher syndrome type 1C caused by the USH1C 216G>A mutation, is treatable using intraperitoneal administration of an ASO.14 ASOs are currently being tested in a number of clinical trials as treatments for muscular dystrophy, Crohn’s disease, and others.47,48 Lentz et al. selected the most effective ASO for correcting splicing mutations from several candidates via in vitro studies. ASOs were intraperitoneally injected into P3-P5 Ush1c216AA knock-in mice, and their auditory and vestibular functions were assessed at 1, 2 and 3 months of age. While control knock-in mice that were administered a scrambled ASO showed profound hearing loss and vestibular dysfunction, mice that received an effective ASO showed significant amelioration of auditory function at both low and mid frequencies, as well as of vestibular functions. Therefore, ASO treatment appears to be a very promising method for congenital hearing loss caused by splicing mutations. However,
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there are several issues that must be clarified and others that need to be resolved. First, we do not know the mechanism by which the intraperitoneally-injected ASOs are transmitted past both the blood-labyrinthine and blood-cochlea barriers into the inner ears. Second, we need to develop better curative treatments, since ASO treatment at P3-P5 demonstrated no amelioration at high frequencies.14 It has also been reported that harmonin, which is encoded by Ush1c, is detectable by E15 at the basal turn of the cochlea and is detectable at the apical turn as late as P30. 49 The absence of amelioration at high frequencies observed in Lentz et al.’s study may therefore be attributable to belated treatments following “normal harmonin initial gene expression” at the basal turn. In addition, to achieve better curative treatments utilizing ASOs, we should consider that the development and maturation of auditory functions in rodents occur later than do those in humans.25,26 Therefore, to achieve more curative treatments, the treatments should be performed at earlier stages, such as the embryonic stage.14
7.
Future directions
The above results regarding congenital genetic hearing loss suggest that normal gene redeeming treatments initiated prior to the “normal gene initiation time” are probably most effective at preventing subsequent hearing loss. Additionally, after the “normal gene initiation time”, the treatment efficacy probably diminishes significantly.42 However, the efficacy of normal gene
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redeeming therapy after the “normal gene initiation time” might vary depending on the causative genes.13 Considering the results of Lentz et al.,14 intraperitoneal administration of an embryonic ASO may be a simple and effective method to treat genetic SNHL caused by splicing mutations. However, wild-type gene supplementation during the embryonic stage is critical for curative treatment of the majority of genetic SNHL cases caused by loss-of-function mutations. To achieve this, we need to clarify and resolve several issues. First, there are limited methods available that can achieve gene transfer in embryonic inner ears at specific periods and locations. New transfer methods satisfying such conditions are necessary. Second, although access to the developing inner ear is necessary to achieve gene transfer, currently the only embryonic stage at which we can access developing mouse inner ears is E11.5 due to anatomical location. There are no reported methods that have accessed developing human inner ears successfully. Therefore, there is an urgent need to develop reliable and safe methods to manipulate developing inner ears at any stage, in both mice and humans. An ultrasound imaging system would likely be useful for this purpose (Fig. 5). Third, treatable time windows and essential locations of curative treatments for each gene have yet to be determined. Additionally, we have summarized the relationship between normal gene expression time in the cochlea and efficacy of normal gene redeeming therapy from the data from previous reports (Fig. 3). However, as mentioned above, Li et al.’s data46 suggest
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that Slc26a4 expression in the cochlea might not be essential for curative treatment. We need to clarify precisely the relationship between the locations essential for curative treatments and treatment timing. Fourth, to perform embryonic treatment, we must be able to precisely diagnose genetic hearing loss during the early embryonic stages. In humans, the otocysts are generated at five weeks gestation. Therefore, to perform embryonic treatment, precise information on the patient’s genetic mutations and functional outcomes caused by the genetic mutation must be obtained at the earliest gestational stage possible. Genetic testing using amniotic fluid, which currently is being performed clinically, would be highly useful for this purpose. In addition, it has been reported that foetal cell-free DNA can be isolated from maternal blood as early as the fifth week of gestation.50 Genetic testing using maternal blood could also be an attractive option for early genetic diagnosis and treatments in the future.51 There is a ray of hope for genetic hearing loss treatments, despite the many issues to overcome. Accumulating knowledge on the cellular and molecular mechanisms involved in inner ear function and morphology, in both mice and humans, will be important to develop clinically-relevant treatments.52 Although we did not discuss treatments utilizing stem cells, they are likely the best option for genetic SNHL treatment in patients following phenotypic manifestation.
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The authors have no conflict of interest directly relevant to the content of this article.
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Figure Legends Figure 1. Transverse section image of the cochlea. The adult mammalian cochlea was divided into three compartments: the scala vestibule, scala tympani and scala media. This image represents a cross-section of the scala media, which contains the organ of Corti (OC). The OC contains three cell populations: inner hair cells (IHCs), outer hair cells (OHCs) and supporting cells. The two types of auditory hair cells (IHCs and OHCs) play critical roles as mechano-electrical transducers for hearing. Auditory hair cells are covered by the tectorial membrane. The stria vascularis, located in the lateral wall of the scala media, is responsible for the secretion of K+ into the endolymph and for production of the endocochlear potential.
Figure 2. To treat genetic hearing loss caused by loss-of-function mutations, there are three important time points: (1) the target gene initiation point at which target gene expression begins in normal individuals (“normal gene initiation time”), (2) the time point at which the normal gene expression matures (“normal gene mature expression time”), and (3) the time point at which phenotypes begin to manifest after target gene deficiency. Miwa et al. demonstrated that otocystic gene transfer before the “normal gene initiation time” can prevent subsequent hearing loss in Cx30-deficient mice. Yu et al. reported that hearing loss in conditional Cx26-knockout mice was untreatable by AAV vector-based gene therapy in the
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neonatal period, which may be after the “normal gene mature expression time” but before phenotype manifestation. Akil et al. reported that AAV-mediated VGLUT3 gene transfer at P1–P12, which corresponds to the time after initial VGLUT3 gene expression in wild type mice and probably also corresponds to the time around or after onset of the hearing loss phenotype manifestation, is effective at restoring hearing in VGULT3-deficient mice. Choi et al. reported that forced expression of Slc26a4 before the “normal gene initiation time” completely prevented subsequent hearing loss, which was diminished after the “normal gene initiation time”. The solid blue lines with arrows indicate successful functional recovery treatment time windows; the solid orange line with arrows indicates a period during which treatment is partially effective. The dotted black lines with arrows indicate the periods during which treatments are ineffective.
Figure 3. The embryonic stage is the ideal period for genetic hearing loss treatment. Miwa et al. reported successful treatment via transuterine gene transfer into the embryonic inner ear in Cx30-knockout mice. Gene transfer was performed by embryonic gene transfer into the developing inner ear in Cx30-deficient mice, which successfully prevented subsequent hearing loss phenotypic manifestation. Electroporation-mediated transuterine gene transfer was performed in otocysts (EUGO) in Cx30-deficient mice at E11.5. Embryos were delivered via
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C-section at E18.5, and the pups that underwent gene transfer at E11.5 were passed to surrogate dams to raise said embryos; these pups did not demonstrate hearing loss at P30.
Figure 4. Choi et al. reported that temporal expression of doxycycline-inducible solute carrier family 26 (Slc26a4) at E0-E16.5 in transgenic mice prevented subsequent hearing loss. The line in the figure indicates the relationship between onset of forced Slc26a4 expression and the consequent hearing threshold at one month post-birth. The efficacy of forced expression for preventing putative postnatal hearing loss decreased rapidly after E16.5, at which Slc26a4 expression begins at the basal turn of the cochlea.
Figure 5. a) Sagittal section of E14.5 mouse embryos in the uterus, observed via a small animal ultrasound imaging system with spatial resolution up to 30 µm (Prospect, S-Shape Corporation, New Taipei, Taiwan). The outline of the embryo is clearly detectable. The asterisk indicates the mouth. Arrowheads indicate the uterine wall. The dotted line indicates the plane on which the embryo’s head was observed in Figure 5b. b) Horizontal section images of the head of an E14.5 mouse embryo in the uterus. The arrow indicates the eye. The asterisk indicates the auricle. The circled area represents the cochlea. The inset image in the right corner shows a magnified image of the cochlea; arrow heads indicate the cochlea. Thus, detection of the cochlea utilizing the
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ultrasound imaging system may be feasible, but the approach to the developing inner ear does not appear to be facile.
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