Advances in Molecular and Cellular Therapies for Hearing Loss

review

© The American Society of Gene Therapy

Advances in Molecular and Cellular Therapies for Hearing Loss Michael S Hildebrand1, Stephen S Newton1,2, Samuel P Gubbels1, Abraham M Sheffield1,3, Amit Kochhar1, Michelle G de Silva4,5, Hans-Henrik M Dahl5,6, Scott D Rose7, Mark A Behlke7 and Richard JH Smith1,3 Department of Otolaryngology—Head and Neck Surgery, University of Iowa, Iowa City, Iowa, USA; 2Program in Gene Therapy, Department of Internal Medicine, University of Iowa, Iowa City, Iowa, USA; 3Interdepartmental PhD Program in Genetics, University of Iowa, Iowa City, Iowa, USA; 4 Particulate Fluids Processing Centre, Department of Chemical and Biomolecular Engineering, University of Melbourne, Victoria, Australia; 5 Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Australia; 6Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Melbourne, Australia; 7Integrated DNA Technologies, Coralville, Iowa, USA 1

Development of effective therapeutics for hearing loss has proven to be a slow and difficult process, evidenced by the lack of restorative medicines and technologies currently available to the otolaryngologist. In large part this is attributable to the limited regenerative potential in cochlear cells and the secondary degeneration of the cochlear architecture that commonly follows sensorineural hearing impairment. Therapeutic advances have been made using animal models, particularly in regeneration and remodeling of spiral ganglion neurons, which retract and die following hair cell loss. Natural regeneration in avian and reptilian systems provides hope that replacement of hair cells is achievable in humans. The most exciting recent advancements in this field have been made in the relatively new areas of cellular replacement and gene therapy. In this review we discuss recent developments in gene- and cell-based therapy for hearing loss, including detailed analysis of therapeutic mechanisms such as RNA interference and stem cell transplantation, as well as in utero delivery to the mammalian inner ear. We explore the advantages and limitations associated with the use of these strategies for inner ear restoration. Received 20 June 2007; accepted 10 October 2007; published online 27 November 2007. doi:10.1038/sj.mt.6300351

Introduction Hearing loss is the most common sensory defect in developed countries.1,2 It is an etiologically heterogenous trait with many known genetic (inherited factors) and environmental (infections, noise exposure, premature birth, exposure to ototoxic drugs) causes. Hearing loss is usually classified as conductive hearing loss or sensorineural hearing loss (SNHL), and can occur in isolation (nonsyndromic) or be associated with other symptoms (syndromic). Conductive impairment can be caused by otitis media, otosclerosis or the presence of a foreign body or tumor and is often reversible, while SNHL involves damage to or loss of hair cells or auditory neurons and is usually irreversible. At least 1 child in 1,000 is born with SNHL of at least 40 dB, including 4 profoundly deaf infants per 10,000.3–6 This kind of hearing impairment has an enormous impact on educational attainment, the use of healthcare systems, the likelihood of employment, and life expectancy. Sixty percent (60%) of congenital and early-onset nonsyndromic hearing loss has a Mendelian basis, with the usual mode of inheritance for these monogenic disorders being autosomal recessive transmission. Presbyacusis (age-related hearing loss) is also highly prevalent in developed societies, affecting 25–40% of individuals >65 years of age.7 Despite the fact that this type of hearing

loss is an increasingly important problem in our aging population, it remains poorly understood. Recessive deafness is most commonly congenital and profound, and accounts for ~80% of all hearing impairment. By contrast, dominant deafness accounts for ~15% of inherited hearing loss and tends to be late-onset and progressive. For both types of hearing impairment hearing aids can provide significant benefits to patients, although with severe-to-profound hearing loss cochlear implantation is the better therapeutic option. It is estimated that the mutation of any of several hundred genes can result in deafness, and to date 21 genes for dominant deafness and 23 genes for recessive deafness have been identified. A further 33 loci for dominant deafness and 44 loci for recessive deafness have been mapped to chromosomal regions.8 The large number of mouse models of human hereditary deafness that have been generated represent a broad spectrum of inner ear defects (reviewed in ref. 9). For example, models of hair cell defects include Shaker-1 (sh; MYO7A) and Waltzer (v; CDH23); those associated with nonsensory cells include Cx26Otog-Cre (GJB2) and Pds–/– (SLC26A4), and those involving defects in the tectorial membrane include Col11a2–/– (COL11A2) and Tecta–/– (TECTA). Because the murine inner ear anatomy and physiology

Correspondence: Michael Hildebrand, Department of Otolaryngology — Head and Neck Surgery, University of Iowa, 5270 CBRB, Iowa City, Iowa 52242, USA. E-mail: [email protected]

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are similar, and in many cases the human deafness phenotype is faithfully recapitulated in the mouse, these mutants provide excellent experimental models to test gene- and cell-based therapies for different forms of hearing impairment.

Gene Therapy RNA interference Identification and development of drug-based therapeutics for otological disorders is costly and time-consuming, and pharmaceutical agents are generally not effective at restoring auditory function. The discovery of RNA interference (RNAi)-­mediated gene inactivation by Fire and Mello10 has introduced a new mechanism for targeted therapy of the inner ear at the molecular level. Sequence-specific RNA knockdown is not a novel concept as both ribozyme and antisense oligonucleotide technologies have been extensively investigated for their therapeutic potential. However, technical issues concerning stability, delivery, target selection, off-target effects, and immune activation have limited their success. The major advantage of RNAi over these traditional approaches is that it makes use of a natural and highly conserved biological process that is present in plants, yeast, invertebrates, and mammals. In plants, RNAi is known to protect cells from viral infection and inappropriate expression of repetitive sequences and transposable elements,11,12 functions that are maintained in mammalian cells.13–15 Recently, a new class of short RNAs was identified in Caenorhabditis elegans and subsequently shown to be present in mammals. These microRNAs regulate a variety of cellular processes including apoptosis, differentiation, and proliferation.16 RNAi is an intracellular two-step process that converts precursor double-stranded RNA molecules into functional small interfering RNAs (siRNAs) (Figure 1). Pre-siRNAs are first processed by the endonuclease Dicer into 21–23 nucleotide fragments with 2 nucleotide single-stranded 3′ overhangs. These fragments are then incorporated into the RNA-inducing silencing complex that Endogenous pathway

Exogenous pathways

N

Genome miRNA biogenesis pol II or pol III transcription Intron or exon RNA Hairpin formation pri-miRNA shRNA Drosha vector uc pre-miRNA l e to u s pl as Exportin 5/Ran GTPase m

Cy

pre-siRNA

siRNA

Dicer Strand incorporation

RISC

High complementarity Degradation

miRNA binding to target mRNAs Transcript 3′ UTR Low complementarity Translational silencing

Figure 1  RNA interference (RNAi) pathways. Endogenous micro RNA (miRNA) and exogenous small interfering RNA (siRNA) pathways for gene silencing. The siRNA pathways utilize the cellular machinery of endogenous miRNA biogenesis and induce degradation of target messenger RNAs (mRNAs). miRNA biogenesis results predominantly in translational repression of target genes and in some cases degradation of target mRNAs. Adapted from Renne R (2007; www.mgm.ufl.edu/faculty/ rrenne.htm). GTPase, guanosine triphosphatase; RISC, RNA-inducing silencing complex; shRNA, short hairpin RNA; UTR, untranslated region.

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unwinds the duplexes and employs one strand, the “guide strand”, to direct sequence-specific cleavage of complementary RNAs.12 Natural siRNAs are generated by the endogenous Dicer processing pathway; alternatively, synthetic double-stranded RNAs can be introduced as siRNA mimics and used to trigger RNAi and intentionally reduce expression of targeted genes for research or therapeutic applications.

Benefits of RNAi therapeutics Potency. The potency of RNAi-mediated gene suppression is typically superior to the knockdown achieved using antisense oligonucleotides. For example, separate studies have shown that siRNAs generated to target sites previously optimized for antisense oligonucleotide selection achieve greater levels of ­inhibition at lower doses under comparably optimal transfection conditions.17,18 These results are consistent with experiments by Elbashir and colleagues showing that antisense oligonucleotide–mediated suppression designed to a target site, optimized for a siRNA, is inferior to suppression achieved by the siRNA.19 However high potency antisense sites with comparable performance to siRNAs do exist.20 That they are rare and more difficult to identify may reflect the fact that RNAi invokes a natural gene regulatory pathway. This versatility of RNAi and its exquisite sensitivity at low doses makes the use of siRNAs as therapeutic agents very ­attractive ­(reviewed in ref. 21). Specificity and versatility. To direct messenger RNA cleavage by RNA-inducing silencing complex, siRNAs must have perfect or near-perfect complementarity to the targeted sequence. Since RNAi-mediated knockdown is based upon nucleic acid hybridi­ zation, theoretically any gene can be targeted, independent of the structure, function, or subcellular localization of the encoded protein. Thus targets previously considered “undruggable” can now be tested for therapeutic value. The identification of effective RNAi target sites is relatively straightforward and potent siRNAs usually can be identified for a desired gene target by testing a relatively small number of candidate duplexes, aided by the use of rational design approaches. Design features that favor high potency siRNAs have been elucidated in a number of studies by different groups and have been summarized in recent reviews.22,23 By contrast, identification of potent sites for antisense oligonucleotides usually requires more intensive ­screening efforts. The specificity of siRNAs makes them very attractive agents for targeting dominant-negative mutations that affect single alleles. The selective knockdown of defective genes without affecting the wild-type copy, has the potential to ameliorate the phenotypic effects of a significant number of genetic, oncological and neurological disorders for which dominant-negative pathological mechanisms are commonly observed.24–27 The feasibility of this degree of specificity has been explored by systematically examining the effects that single base changes have on functional knockdown. In one study, mutations in the target sequence were examined while the siRNA was kept constant,28 while in another study, the reverse was done by varying the siRNA sequence instead.29 These reports provide useful guidelines for the design of allele-specific siRNA reagents. 225

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Gene therapy and viral vectors In the last decade a series of gene therapy studies targeted at the inner ear have been reported. The most promising advance in the application of gene therapy to restore auditory function has been the discovery that Atoh1 (Math1) induces hair cell differentiation.30–32 Izumikawa and colleagues showed that transfer of adenoviral (AV) vectors expressing Math1 resulted in the formation of “hair cell–like” cells in the guinea pig organ of Corti 5 weeks postinoculation.33 In 2 months, the surface of the auditory epithelium contained numerous cells with mature-looking stereocilia bundles: cross sections of the organ of Corti revealed inner hair cells (IHCs) with normal morphology. New outer hair cells, however, remained poorly differentiated suggesting that additional factors are required to specify outer hair cell development. Functional testing at 2 months postinoculation revealed significantly improved auditory brainstem responses consistent with Math1-induced regeneration of “hair cell–like” cells.33 This study also showed that in addition to being effective, the in vivo delivery of AV was safe. The variety of viral and nonviral vectors available for delivery of therapeutic RNAs or genes to target cells in the inner ear is large. Viral vectors offer the advantage of sustained expression in cochlear cells, with the choice of vector being dependent upon virus-specific properties. The properties of the most widely used viral vectors for gene therapy are described in Table 1 and below. The application of these vectors in gene therapy approaches to the inner ear is summarized in Table 2.

AV. A relatively benign human pathogen, AV infection causes the common cold or conjunctivitis. However, replication-­deficient AV is considered safe.34 A major advantage of AV vectors compared to most retroviral vectors is their ability to infect both dividing and nondividing cells with high efficiency. These vectors also have a broad host range and short time of onset following infection, making them appropriate for a wide variety of targets. In the inner ear, a diverse group of cell types can be successfully transduced by AV, including sensory and nonsensory cells of the organ of Corti, fibrocytes in the spiral ligament and spiral ganglion neurons (SGNs). However, significant problems associated with the use of AV include transient expression due to failure of the virus to integrate into the target genome and induction of a potent immune response that may result in toxicity. Adeno-associated virus. This member of the parvoviridae family is the smallest known human virus and is yet to be linked to any human pathology.35 Unlike AV, adeno-associated virus (AAV) readily integrates into the host genome, permitting longterm expression of a transgene. Malignant transformation has not been associated with genome integration and there have been no reports of significant immune response to AAV infection. A large number of cell types in the inner ear can be targeted with AAV, including inner and outer hair cells, supporting cells, and SGNs. The most efficient transduction of cochlear cells has been ­reported for the AAV2/1 serotype.36 A major caveat for the use

Table 1 Properties of viral vectors commonly utilized for in vivo gene delivery Vector

Adenovirus (AV)

Adeno-associated virus (AAV)

Lentivirus (LV)

Family

Adenoviridae

Parvoviridae

Retroviridae

Size

70–90 nm

20–25 nm

100 nm

Type

Double-stranded DNA

Single-stranded DNA

RNA

Packaging capacity

Medium <7.5 kilobase (kb)

Low <4 kb

Medium 8 kb

Enveloped

No

No

Yes

Host range

Broad

Broad, dividing/nondividing cells

Broad, dividing/nondividing cells

Transduction efficiency

Low

High

Low

Time of onset

2 days

0–4 weeks

4 days

Expression

Transient

Long-term, slow onset

Long-term

Genome integration

No

Yes, rare

Yes

Immunogenicity

High

Low

Low

Production efficiency

High

Low

Low

Safety

Medium

Medium

Low

Clinical trials

Yes

Yes

No

Clinical applications

Tumors Hematopoietic cells

Liver, muscle, CNS Retinal cells

CNS, liver, muscle

Ototoxicity

No

No

Yes

Serotype used in cochlea

AV5

AAV2/1, 2/2, 2/5, 2/6, 2/7, 2/8, 2/9, 3

VSVG-HIV

Cochlear cell types transduced (in vivo)

Hair cells Supporting cells Spiral ganglion neurons Fibrocytes (spiral ligament, perilymphatic) Stria vascularis Reissner’s membrane

Outer hair cells Inner hair cells Supporting cells Spiral limbus Spiral ganglion neurons Reissner’s membrane Spiral ligament

Spiral ganglion neurons Fibrocytes (spiral ligament, perilymphatic) Reissner’s membrane

Abbreviations: CNS, central nervous system; HIV, human immunodeficiency virus; VSVG, Vesicular stomatitis virus glycoprotein.

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Table 2  Gene therapy experiments targeted at the inner ear Gene

Target Cell

Vehicle

Recipient species

Delivery route

Reference

GDNF

Spiral ganglion neurons

Ad

Guinea pig

Scala tympani

79

HGF

Spiral ganglion neurons

HVJ-E

Rat

Intrathecal

80

Catalase

Hair cells

Ad

Guinea pig

Scala media

90

LacZ

Hair cells

Ad

Guinea pig

Scala tympani

BDNF

Spiral ganglion neurons

Ad

Guinea pig

Scala tympani

NT3

Hair cells

Liposome

Guinea pig

Scala media

Atoh1

Supporting cells

Ad

Guinea pig

Scala media

EGFP

Inner hair cells

AAV

Mouse

Scala tympani

130

GJB2

Supporting cells spiral ligament

Liposome

Mouse

Scala tympani

60

Myo15a

Hair cells

Crossing

Mouse

Germline

65

EGFP

Hair cells

AAV lentivirus

Mouse

Otocyst

36

BDNF

Spiral ganglion neurons

Fibroblast cell lipofectamine

Mouse

Semicircular canals

82

BDNF

Spiral ganglion neurons

Fibroblast cell Ad

Guinea pig

Scala tympani

83

  SOD1   SOD2 128 81 129 33

Abbreviations: AAV, adeno-associated virus; BDNF, brain-derived neurotrophic factor; EGFP, enhanced green fluorescent protein; GDNF, glial cell line-derived neurotrophic factor; HGF, hepatocyte growth factor.

of AAV in transgene delivery is its relatively small packaging capacity (~4.5 kilobase), limiting the types of genes that can be delivered. Lentivirus. A vector derived from the human immunodeficiency virus, lentivirus (LV) is unique among retroviruses in its ability to infect dividing, nondividing and mitotically inactive cells.37 LVs readily integrate into the genome providing long-term gene ­expression even in nonproliferating, postmitotic cells. Since a number of cochlear cell types are postmitotic, including SGNs, these cells are good targets for LV infection. A relatively large packaging capacity and broad host range also make LV attractive for gene therapy applications. The major downsides to the use of LV in the inner ear include high immunogenicity and ototoxicity.36

Delivery routes The most commonly used routes for intracochlear delivery of vectors involve accessing the scala tympani or the endolymphatic sac (Table 2). Accessing the scala tympani is technically easy with little chance of iatrogenic trauma, and while hearing impairment associated with penetration of the perilymphatic compartment can occur, it is not inevitably permanent.38 By applying Gelfoam soaked in therapeutic agents to the round window membrane (RWM) the need to directly access the cochlea is obviated.39,40 However, diffusion across the RWM is vector-dependent, and while liposomes and some viral vectors can passively diffuse across this barrier, AAV serotypes cannot.39 Efficient viral transduction of mature cochlear hair cells may be challenging as a number of studies using AV, AAV, and LV have reported little or no transduction of these cells.39,41 This could be an issue of physical access as transplanted cells are likely to be unable to access the hair cells from the scala media or scala tympani. Only the apical surface of the hair cells face into the scala media with their distinctive stereocilia ­projecting through Molecular Therapy vol. 16 no. 2 feb. 2008

the cuticular plate. The true division between the endolymphatic and perilymphatic compartments is the reticular lamina which provides a tight junction barrier that restricts the interchange of materials between these regions.42 It is therefore unclear whether viral agents would be able to penetrate the reticular lamina to reach the hair cells. However, it is known that migration is possible through tight junction barriers, such as binucleate cells that transverse the ionic barrier seal, the trophectodermal epithelium, throughout pregnancy in the ruminant placenta.43,44 Likewise, access to the organ of Corti from the scala tympani is likely to be restricted by the presence of the basilar membrane, potentially preventing transduction by virus admini­stered via this route. The lack of transduction of mature hair cells may also suggest that the appropriate viral receptors, such as the coxsackievirus and adenovirus receptor and integrins avb5 and avb3 for AV, and heparin sulfate for AAV, may be present in only low numbers or are absent form these cells.45–47 As an alternative, Bedrosian and colleagues targeted progenitor cells in the otocyst of the embryonic day (E) 12–12.5 mouse embryo that give rise to the hair and supporting cells.36 A laparotomy is performed on a dam at the appropriate gestational stage and the therapeutic agent housed in viral vector is delivered directly into the otocyst via ex utero or trans-uterine microinjection (Figure 2). Highly efficient AAV transduction of progenitor cells is observed, with postnatal transduction percentages of ~80 and ~65% for inner and outer hair cells, respectively.36 Transgene expression persists for >6 months, ensuring a wide therapeutic window and the potential for treatment of progressive forms of hearing loss. Bedrosian and colleagues also reported no detrimental effects on auditory function in mice injected with the AAV construct. The increased transduction of progenitor cells may reflect differences in the viral receptors on the surface of these cells compared to mature hair cells. 227

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a

© The American Society of Gene Therapy

b

IV V

c

V

d IV

Figure 2  Ex utero microinjection into the developing mouse otocyst. The ex utero approach as seen through the surgical microscope. (a) The uterus is exposed by a midline laparotomy and the fetuses appear as a string of beads. The uterine wall (arrow) is incised to expose the fetus. (b) The microinjection pipette (arrow) enters the amniotic sac. The triangular injection site is demarked by the cardinal veins (v) and fourth ventricle (IV). (c) Dorsal view of the embryo at embryonic day 12 (E12). The left eye (arrow) and fourth ventricle are visible. Following injection, fast green dye is visible in the otocyst indicating successful injection. (d) Left sagittal profile of the embryo illustrating the endolymphatic duct (arrow) protruding away from the otocyst at the 1 o’clock position.

Safety concerns The ability to administer therapeutic agents topically to the inner ear avoids many of the safety concerns associated with systemic delivery. The blood–labyrinth barrier ensures stability of the unique extracellular cochlear fluid compartments by providing selective permeability48 and also makes the cochlea a relatively immunoprotected site.49,50 Studies of the C3H/lpr autoimmune mouse, for example, show that during active disease, hearing loss is accompanied by breakdown of the blood–labyrinth barrier.49 Local off-target effects induced by the therapeutic agent and nonspecific effects provoked by the agent and/or vector, however, remain serious risks of gene therapy. Off-target effects relate to the nature of the delivered gene or RNA sequence and the degree of homology to other endogenous genes. These effects can be minimized by rigorous screening of potential genetic or siRNA targets, and by selective chemical modification of the siRNA. Toxicity associated with nonspecific effects is more difficult to avoid, as both the delivery vehicle and the therapeutic agent can be toxic or trigger an innate immune response. The nucleic acid molecules themselves can be recognized as “foreign” and trigger an interferon response. For example, Toll-like receptor 9 (TLR9) recognizes nonmethylated dC bases in the context of a “CpG” dinucleotide motif in DNA antisense oligonucleotides or plasmids, and a large number of immune receptors recognize RNA, including TLR3, TLR7, TLR8 and the retinoic inducible gene-1 (RIG-I) (reviewed in refs. 51,52). Responses are both sequence and cell-type specific and therefore are not inevitably observed in cell culture. Nevertheless, they remain a potential concern in vivo where all cell types are present. 2′-O-methyl RNA chemical modification of rU and rG residues in siRNAs can eliminate many of these nonspecific effects.53 Viral vectors also trigger an innate immune response, making the degree of immunogenicity an important consideration in 228

v­ ector selection. Given the proximity of the central nervous system, dissemination of virus outside the targeted inner ear structures is a concern. In this regard, transgene expression within the contralateral cochlea has been demonstrated using both AV and AAV.54,55 The likely route of migration of these viruses is through the cerebrospinal fluid or bone marrow of the temporal bone. Microinjection or RWM application of these and other viruses avoids this dissemination.39,56

Gene therapy for hereditary hearing loss Approximately 15% of hereditary deafness is inherited as autosomal dominant nonsyndromic hearing loss, and in many instances, the deafness phenotype reflects a dominant-negative mechanism of action. Examples include GJB3 (DFNA2),57 GJB2 (DFNA3),58 GJB6 (DFNA3),59 and possibly also DFNA4, DFNA9, and DFNA20/26. Since inheritance is autosomal dominant, silencing of the mutated allele would be predicted to preserve hearing. A recent proof-of-principle study validated this prediction—a siRNA was shown to potently suppress expression of the R75W allele of human GJB2 in a murine model.60 By using a construct ­containing GJB2R75W that interferes with the functioning of the wild-type gap junction protein,61 Maeda and colleagues were able to recapitulate human deafness (DFNA3) in a murine model.60 In subsequent experiments, the same construct and specific anti-GJB2R75W siRNAs were mixed with 1,2-dioleoyl-3-trimethylammonium propane/ cholesterol liposomes, soaked in Gelfoam, and applied topically to the murine RWM. Although liposome–nucleic acid complexes were detected in nonsensory cells of the cochlea, the siRNA specifically reduced expression of the GJB2R75W allele and prevented the hearing loss phenotype.60 A number of recent studies report the knockdown of other inner ear genes in vitro and in vivo. However, none of these genes have been implicated in human deafness. Yamanishi and colleagues reported siRNA-induced knockdown of the bone morphogenetic protein antagonist gene Dan during chick inner ear development that resulted in reduction in the size of the endolymphatic duct and sac, and malformations of the semicircular canals and cochlear duct.62 Two studies from the laboratory of R-K Park describe reduced expression of the heme oxygenase-1 gene (HO-1), implicated in protection against cisplatin toxicity, following treatment of an auditory cell line with a targeted siRNA.63,64 Knockdown of HO-1 significantly reduced the viablity of the auditory cells in the presence of cisplatin. Based on the results achieved with GJB2, it is highly likely that alleles of other genes that cause autosomal dominant nonsyndromic hearing loss can be targeted by RNAi therapy delivered at different developmental time-points through different surgical approaches. Using a murine model, one example involves the delivery of an AAV vector to the E12.5 mouse otocyst via a transuterine approach36 prior to the onset of hearing loss (­Figure 2). In this approach, progenitor cells that give rise to the tissues of the mature inner ear are transduced with a therapeutic agent. Alternatively, the RWM of the adult mouse cochlea can be targeted via a ventro-medial approach.39,40 Cell-specificity with either approach requires further study as current delivery systems may limit the applicability of these approaches to specific types of hearing loss. www.moleculartherapy.org vol. 16 no. 2 feb. 2008

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While transgene expression in the adult organism has the ability to restore auditory function, gene augmentation in the germline can be used to prevent hearing loss by maintaining normal inner ear morphology. This approach can potentially be applied to treat recessive forms of deafness that constitute the most common type of hearing impairment. Recently, transgene expression through the germline was demonstrated to maintain inner ear morphology and hearing function in a mouse model of human nonsyndromic deafness DFNB3.65 Kanzaki and colleagues introduced a bacterial artificial chromosome (BAC) transgene containing wild-type Myo15a to the shaker2 (sh2) mouse model that exhibits profound, congenital SNHL due to mutations in the endogenous copies of this gene. Transgene expression in these mice up to 6 months of age resulted in normal hearing function and abolished circling behavior. Morphological examination revealed normal numbers and architecture of hair cells in the apical and upper basal turns.65 Since transgene expression can vary significantly depending on copy number and endogenous influences, the effect of BAC-Myo15a expression was evaluated in phenotypically normal sh2 heterozygotes. No deleterious effects of excess transgene ­expression were observed suggesting that clinical therapy using Myo15a would be safe. A difficulty in developing clinical therapies for recessive forms of deafness such as DFNB3 is being able to intervene at the appropriate stage. Intervention prior to development of hair cells or during the early postnatal period when hair cells may still be intact will be necessary to prevent the pathological changes that generally occur congenitally in recessively inherited forms of hearing loss.

Neurotrophic factors The loss of IHCs in sensorineural deafness is usually followed by degeneration of SGNs. The rate and extent of SGN degeneration can be quite variable as evidenced by both animal and clinical studies.66–68 The degeneration of SGNs is attributed to a loss of survival factors such as neurotrophins and the dopolarizing activity of IHCs.69,70 While cochlear implantation can provide sensory function for individuals with profound hearing impairment, the effectiveness of the implant is dependent on both the residual function and number of surviving SGNs.71–73 Since individuals with profound deafness generally do not receive cochlear implants until at least 6 months after deafness, there is a significant window during which therapeutic intervention with survival factors such as neurotrophins or chronic stimulation could reduce SGN degeneration. Preservation of SGNs during this period would lead to improved outcomes for cochlear implant recipients. The developing and mature cochlea contains neurotrophins and their receptors that play crucial roles in the development of the sensory epithelium in the cochlea and vestibule (reviewed in ref. 74). Neurotrophins have been shown to excite SGNs and in the absence of normal stimulation can enhance their survival.69 One of the neurotrophins expressed in the inner ear is brain-derived neurotrophic factor (BDNF) and it has been demonstrated to have a significant role in cochlear development.75 Supplementation of exogenous BDNF directly into the cochlea following IHC loss enhances SGN survival.76–78 One problem with inner ear delivery of factors such as BDNF via a ­ miniosmotic pump system or bolus injection is rapid degradation Molecular Therapy vol. 16 no. 2 feb. 2008

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and short-term ­function. An ­alternative to these approaches that has been examined experimentally is delivery of neurotrophic genes to the inner ear that results in expression of the gene product by endogenous cells. Secretion of proteins from glial cell line-derived neurotrophic factor,79 hepatocyte growth factor,80 and BDNF81–83 transgenes has been reported in the mammalian inner ear and has been shown to preserve SGNs following ototoxic damage (Table 2). In the case of hepatocyte growth factor introduction to the rat cochlear, a protective effect was also observed for IHCs.80 The protective effect of BDNF on SGNs is well-established and several gene therapy studies have reported introduction of this neurotrophin to the mammalian inner ear.81–83 Nakaizumi and colleagues used AV to deliver mouse BDNF and rat ciliary– derived neurotrophic factor (CNTF) into the scala tympani of the guinea pig inner ear.81 CNTF is known to promote survival of hippocampal neurons and its receptors are also expressed in the inner ear. Hair cells were eliminated in the guinea pigs using kanamycin 7 days prior to the transgene delivery and only animals with auditory brainstem response thresholds >60 dB were included in the study. Spiral ganglion histology was analyzed 28 days postdeafening to compare SGN density. Comparison of cochleae treated with Ad.BDNF, Ad.CNTF or both to Ad.null, artificial perilymph or nondeafened controls revealed robust SGN survival in those cochleae that received Ad.BDNF alone or in combination with CNTF. The survival was most significant in the basal and second turns, and was equally distributed around Rosenthal’s canal. However, treatment with CNTF alone failed to promote survival of SGNs compared to the controls, suggesting that this neurotrophic factor does not have a strong protective effect on these neurons. The strong protective effect of BDNF has been validated by a subsequent study reported from the same laboratory.83 In this study, cochlear implant therapy was combined with ex vivo gene therapy to determine if application of BDNF via a cochlear implant could induce SGN survival. Guinea pig fibroblasts were transduced with a BDNF gene cassette and secretion of the ­protein from these cells confirmed.83 Cochlear implant electrodes were then coated with the BDNF-secreting fibroblasts in an agarose gel and inserted into the scala tympani of a deafened guinea pig model. Histological analysis 48 days postimplantation revealed significantly greater numbers of SGNs in the basal turns of the cochlea receiving the BDNF-secreting implant compared to those receiving the Ad.empty control implant. The outcomes of this study provide evidence that the cochlear implant is a feasible route for delivery of neurotrophic and other therapeutic agents to the mammalian inner ear. In a related and recent study, cell-gene delivery of BDNF to the mouse inner ear was reported.83 In this novel therapeutic approach, Okano and colleagues transplanted NIH3T3 cells that had been engineered in vitro to express BDNF into the posterior semicircular canals of the adult mouse inner ear. Delivery of the BDNF transgene to the NIH3T3 cells was facilitated by nonviral lipofection, avoiding the potential side-effects associated with viral reagents. The cells were shown to survive in the inner ear for up to 4 weeks after transplantation and an enzyme-linked immunosorbent assay confirmed increased expression of BDNF in the inner ear.83 While the functional implications of the elevated BDNF 229

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were not examined in this study, this novel delivery methodology may represent a safe and effective way to deliver neurotrophins to the inner ear.

Antioxidant therapy Administration of glucocorticoids has been shown to have a positive therapeutic effect on sudden SNHL, Meniere’s disease and noise-induced hearing loss.84 This benefit may reflect the effect of glucocorticoids on the regulation of genes associated with ion transport as well as their well known anti-inflammatory and/or immunosuppressive action.85 Indeed, the discovery of new therapies for the prevention or treatment of congenital hearing loss, presbyacusis and noise-induced hearing loss will depend on a better understanding of gene function in maintaining the unique inner ear ion balance. The inner ear is also a highly energy-dependent organ and it is therefore not surprising that near-optimal mitochondrial function is required for normal hearing. Hearing loss is a feature in most syndromes involving mitochondrial dysfunction.86 Highly toxic reactive oxygen species are generated in the mitochondria as a consequence of energy generation and slowly impair mitochondrial function by damaging biological molecules (including the mitochondrial DNA). One consequence is progressive hearing loss.87 Antioxidants to scavenge reactive oxygen species can protect hair cells from noise-induced damage and ototoxicity and preserve auditory function.88–91 Although systemic delivery of antioxidants is effective in protecting the inner ear, efficacy is limited by a lack of tissue specificity, unwanted side effects, a short half-life, and the blood–labyrinth barrier. An alternative, therefore, is gene therapy to over-express antioxidant enzymes in the inner ear. Kawamoto and colleagues reported AV-mediated delivery of catalase and the SOD1 and SOD2 superoxide dismutase genes in a guinea pig model of aminoglycoside ototoxicity.90 The over-expression of SOD1 in a mouse model had also been shown to protect against aminoglycoside-induced hearing loss. The protective effect of Ad.catalase and Ad.SOD2 on function correlated with morphological analyses showing the number, structure, and architecture of IHC and outer hair cells was preserved. This study demonstrated that functional enzymes could be introduced into the cochlea via transgenes and that these enzymes can have a protective effect on structure and function. The implication is that deafness induced by reactive oxygen species is a suitable disease target for gene therapy approaches that modulate expression of antioxidants.

Cell Therapy Cell transplantation and the inner ear A principal focus of contemporary otolaryngology is restoration of function by replacement of damaged tissues or organs. This replacement can be facilitated either by transplantation of tissue or implantation of artificial materials. There are significant caveats associated with both of these approaches, including the propensity for immune rejection, potential donor site morbidity and the limited supply of donor tissue. Effective, stable and long-lasting repair of damaged tissue may require only transplantation of pluripotent progenitor cells; however, in order to achieve complete functional restoration especially in the inner ear, it is probable 230

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that both stem cell transplantation and tissue engineering will be needed to provide the appropriate environment to induce normal differentiation92 (Figure 3). Numerous criteria must be satisfied to achieve functional restoration including generation of an adequate number of cells to reverse the defect, differentiation of cells to the correct phenotype, formation of appropriate three-dimensional tissue structures, ­production of cells/tissues that are mechanically and structurally compliant with the native tissue, and integration of the transplanted tissue with the native tissue without immunological rejection.92 While it has long been possible to isolate tissues for autologous implants and avoid immunological rejection, this technique is currently not practical for therapeutic purposes. The cells generally have low proliferative potential, are invariably restricted to the type of tissue from which they are harvested, and are difficult to obtain in sufficient quantities.92 What constitutes the most efficacious source of stem cells for therapeutic application is debatable. There is no doubt that adult stem cells exist in a far greater number of tissues than was ever predicted, including in the nervous system, dermis, blood, and inner ear, and are much more plastic than previously thought.93 These cells can be maintained in vitro and proliferate for many generations without losing their differentiation potential and then can be induced to differentiate down separate lineages to form osteoblasts, Cell membranes

Connexin Connexon Scala vestibuli (Perilymph) Scala media (Endolymph) Temporal membrane Outer hair cell

Inner hair cell

Bone

Scala tympani (Perilymph)

Bone

Figure 3  Location of connexin 26 (GJB2) in the potassium recycling pathway of the cochlea. Connexin 26 is expressed in the nonsensory epithelial cells (interdental cells of the spiral limbus, inner and outer sulcus cells, sensory supporting cells, and cells within the root process of the spiral ligament) shown in green, and the connective tissue cell system (fibrocytes of the spiral ligament and spiral limbus, basal and intermediate cells of the stria vascularis) depicted in brown. Each connexin 26 molecule is known as a connexin (yellow). Six connexins oligomerise to form a connexon, and each connexon joins with another to form a gap junction. (Adapted from refs. 143,144.)

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chrondocytes, or adipocytes.93 They have the capacity, both in vitro and in vivo, to give rise to cells of all three germ layers. For example, they acquire a neural phenotype when delivered to the rat brain, a myocardial phenotype in the rat heart and a hepatocyte phenotype in the human liver.93 Embryonic stem (ES) cells can be maintained indefinitely as a pluripotent cell population in vitro, retain the ability to respond to the normal signals that regulate mammalian development, allow precise manipulation of the genome, and differentiate into suitable populations for implantation.94 It is therefore crucial that studies focused on stem cell therapy involve investigation of both ES cells and adult stem cells. A number of cell therapy studies have demonstrated s­uccessful delivery of ES and adult stem cells to normal and ­ damaged ­tissues in vivo, and in some cases a therapeutic effect has been observed.95,96 Adult rat hippocampus–derived neural stem cells (NSCs) have been successfully grafted in brain regions such as the hippocampus, olfactory bulb, and retina and once there, have been shown to differentiate down a neural pathway.97–99 An investigation conducted by Bjorklund and colleagues (2002) illustrates the potential of ES cells for therapeutic outcomes. Mouse ES cells were differentiated into embryoid bodies over 4 days in the absence of mouse leukemia inhibitory factor and the cells were then transplanted into the striatum of immunosuppressed rats.96 The ES cells proliferated spontaneously into fully differentiated dopaminergic neurons within the striatum over a period of 14–16 weeks. These neurons induced gradual and sustained behavioral restoration of dopamine-mediated motor asymmetry and re-established cerebral function and behavior in an animal model of Parkinson’s disease.96 The most promising aspects of this study in the context of a similar approach for cell therapy in the inner ear are that the ES cells exhibited low immunogenicity, as they survived and differentiated after cross-species transplantation, and that functional restoration was achieved from progenitor cells. A number of equally encouraging studies have shown that adult bone marrow stem cells can repair and restore damage to tissues from other organs.100–102

process. The transcription factor Math1 is also essential for hair cell development after hair cell precursor selection has been specified during development of the organ of Corti. Hair cells are absent in mice lacking Math1 and in contrast, the over-expression of Math1 causes the production of extra hair cells.30,112 Once hair cells have been specified, their continued differentiation requires the class IV POU-domain gene, Pou4f3. Without the presence of this gene product, the hair cell precursor cells degenerate. One of the first reports of stem cell delivery to the inner ear was a study by Ito and colleagues (2001) that demonstrated survival and migration of adult rat NSCs implanted into the rat cochlea (Table 3). The β-galactosidase (β-Gal)-expressing cells

Cell delivery to the cochlea The aim of cell therapy in the cochlea is to replace damaged or lost cells, particularly sensory hair cells and spiral ganglion cells, without disturbing cochlear architecture and preserving existing hearing function. Inner ear hair cell degeneration and loss induced by loud sound,103 exposure to ototoxic drugs,104 aging or hereditary gene defects is responsible for >80% of all cases of hearing impairment.105–107 Loss of mammalian hair cells damaged by administration of ototoxic drugs or acoustic over stimulation is permanent and causes irreversible hearing defects.104 However, the discovery that hair cells of the avian basilar papilla, the functional equivalent of the mammalian organ of Corti, regenerate108 after pre-existing hair cells have been destroyed has stimulated much interest in the possibility of hair cell regeneration therapy in mammals.109 The reason that regeneration of inner ear hair cells does not occur in the mammalian inner ear110,111 remains unclear. In mammals, hair cells are terminally differentiated cells and the vast majority of them arise before birth. In the mouse, for example, hair and supporting cell proliferation culminates between E13 and 15. The Notch signalling pathway plays a central role with Notch1 and its ligands Delta1, Jagged1, and Jagged2 in regulating this ­complex Molecular Therapy vol. 16 no. 2 feb. 2008

Table 3 Cell therapy experiments targeted at the inner ear Cell Type

Recipient species

Delivery route

Bone marrow cells (mouse)

Mouse

Intravenous

131

Adult neural stem cells (rat)

Rat

Scala tympani

113

Embryonic neural stem cells (mouse)

Mouse

Semicircular canals

124

Reference

Dorsal root ganglia (guinea pig) Guinea pig Scala tympani

132

Fetal neural stem cells (mouse) Mouse

Scala tympani

114

Bone marrow stromal cells (chinchilla)

modiolus

133

Fetal neural stem cells (mouse) mouse

modiolus

134

Otocyst cells (rat)

Rat

Scala tympani

135

Embryonic stem cells (mouse)

mouse

Semicircular canals

115

Dorsal root ganglia (mouse)

Rat

Scala tympani

136

Embryonic stem cells (mouse)

Rat

Auditory nerve fibres

116

Dorsal root ganglia (mouse)

Rat

Scala tympani

137

Dorsal root ganglia (mouse)

Guinea pig/rat

Vestibulocochlear nerve

138

chinchilla

Dorsal root ganglia (mouse)

Embryonic stem cells (mouse) Embryonic stem cells (mouse)

Guinea pig Scala tympani

139

Embryonic stem cells (mouse)

Guinea pig Scala media

117

Embryonic stem cells (mouse)

Guinea pig Scala tympani

140

Neural progenitors (mouse)

Guinea pig Modiolus

141

Embryonic stem cells (mouse)

Rat

Auditory nerve fibres

142

Marrow-derived stem cells (mouse)

Mouse

Intravenous

118

Marrow-derived stem cells (mouse)

Gerbil

Scala tympani/ modiolus

119

Neural progenitors (mouse)

Gerbil

Cochlear nerve trunk

120

Fibroblasts–BDNF (mouse)

Mouse

Semicircular canals

Dorsal root ganglia (mouse)

Haematopoietic stem cells (mouse)

82

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migrated to the organ of Corti where some cells adopted hair celllike morphology.113 However, the number of NSCs adopting this morphology was limited. The authors speculated that if the stem cells could localize to the correct region of the cochlea then they would take on hair cell characteristics.113 However, the correct targeting of stem cells to the organ of Corti alone is unlikely to be sufficient to promote hair-cell development and differentiation as the appropriate developmental cues may not be present in the adult cochlea. Rather, the partial differentiation of ES cells in vitro prior to implantation may provide these cells with the developmental potential to form new hair cells. Following this study was a report about the fate of adult NSCs delivered to the damaged mouse cochlea.114 The cochlear hair cells were destroyed or damaged by local injection of the ototoxic antibiotic neomycin, and NSCs in the form of neurospheres were delivered through a hole made in the second turn of the cochlea. Almost half the transplanted cells migrated to the vestibular epithelium and ~5% expressed the hair cell marker myosin VIIa after 25 days. By contrast, a very small number of stem cells migrated to the cochlear sensory epithelium and none expressed myosin VIIa.114 The results of this study suggest that the loss of hair cells can induce regeneration of hair cells from progenitors, but it is unclear whether this is possible in the cochlear sensory ­epithelium. Other groups have also reported on the transplantation of ES cells into the inner ear.115,116 Sakamoto and colleagues reported survival of ES cells predominantly in the vestibular region of the mouse inner ear and also some cells in the scala media of the cochlear duct after transplantation for 4 weeks. Hu and colleagues demonstrated the survival and migration of mouse ES cells along the auditory nerve after xenotransplantation into

a

auditory nerve fibers of the rat cochlea. They showed that the ES cells could survive for up to 9 weeks and that they migrated along the auditory nerve fibers into the brainstem. While these studies demonstrate the survival of ES cells in the cochlea, the efficiency with which cells are delivered to the scala media has been low and neither study has examined the survival of partially differentiated cell types. Recently, attention has focused not only on the therapeutic potential of ES cells in the damaged inner ear but also adult bone marrow–derived stem cells. Both cell types are available in sufficient supply to enable their therapeutic use, and bone marrow– derived stem cells (MSCs) are not only considered acceptable for transplantation but are already widely used in the clinic. A variety of therapeutic models, including those outlined in Figure 4 (ref. 117) and Table 3, have examined the delivery and therapeutic response of ES cells in various compartments of the mouse, rat, and guinea pig cochlea. MSCs have been delivered both systemically and by direct injection through the scala tympani into the mouse and gerbil cochlea respectively.118,119 Matsuoka and colleagues investigated the potential of MSCs to adopt properties of SGNs in vivo.119 MSCs were obtained from ACTbEGFP mice and injected into the perilymphatic compartment of the adult gerbil cochlea via scala tympani or modiolar injection. Histological analysis conducted 7 days following implantation revealed most enhanced green fluorescent protein–positive transplanted cells were present in the scala tympani and scala vestibuli, and only a few in the scala media. This pattern was observed for both delivery methods; however there were no transplanted cells evident in the modiolus of animals treated using the scala tympani ­injection. The survival rate of the MSCs in the cochlea was comparable to other studies using ES cells and NSCs. Since MSCs are able to

b

BM

RWM

c

d

SV

SL SL

e

SV

SV

SM

SM SM OC

OC ST ST

Figure 4  Delivery of mouse embryonic stem (ES) cells to the deafened guinea pig cochlea. (a) A postauricular approach was used to expose the round window membrane (RWM) and underlying basilar membrane (BM) within the tympanic bulla (×10 magnification). (b) Insertion of a polyimide cannula through the basilar membrane to facilitate direct implantation of mouse ES cells into the scala media (×20 magnification). (c–e) Localization of mouse ES cells in the three scalae of the guinea pig cochlea following injection. The transplanted cells were detected by their expression of enhanced green fluorescent protein and their morphology was determined by histological analysis. Adapted from ref. 145. OC, organ of Corti; SL, spiral limbus; SM, scala media; SV, scala vestibuli; ST, scala tympani; SV, stria vascularis.

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migrate, the outcomes of this study indicate that there is an anatomical barrier between the modiolus and perilymphatic space, and thus further experimentation is required to determine the optimal delivery method to target cells to the spiral ganglion and organ of Corti. While many of the studies listed in Table 3 have demonstrated the delivery and survival of stem cells in the inner ear, few have reported differentiation and subsequent regeneration of endogenous cell types. One study that has elegantly demonstrated stem cell–mediated regeneration of cochlear neurons, was reported recently by Corrales and colleagues.120 Mouse enhanced yellow fluorescent protein–expressing neural progenitors derived from ES cells were injected into the cochlear nerve trunk via the round window niche of adult gerbils. Prior to the transplantation, the gerbils were treated with ouabain applied directly to the round window to destroy most of the cochlear neurons while leaving the hair cells intact. This was confirmed by measurement of normal distortion product otoacoustic emissions that are created by hair cells and elevated compound action potential thresholds that represent cochlear nerve fibre activity.120 Twelve days following transplantation the enhanced yellow fluorescent protein-positive neural progenitors had developed processes that extended toward the denervated organ of Corti. In 64–98 days these neurons had formed bundles through Rosenthal’s canal and the osseous spiral lamina to the organ of Corti where contact was made with hair cells. The survival rate (13.6%) of transplanted cells was higher than previous reports involving delivery of undifferentiated cell types. The remarkable morphological changes demonstrate the capacity of partially differentiated neural progenitors to form neurons that appear to share many properties of cochlear neurons. This kind of predifferentiation and targeted delivery of progenitor cells represents an exciting development in cell-based therapies for hearing loss and the functional analyses of such experimental models will be intriguing.

Transplantation and auditory trauma A number of studies have investigated the amount of trauma sustained during surgical access to the inner ear.113,114,121–124 Separate investigations on the mechanical compliance of the endolymphatic compartments have shown that there is no difference in pressure between the endolymph and perilymph after injection of up to 2 µl of artificial endolymph.121,122 This is significant, especially given that the normal endolymph volume of the guinea pig cochlea is only ~1.2 µl. The injection of substances into the scala media through the stria vascularis does not appear to rupture the membranous labyrinth or cause significant trauma, provided the delivery rate is <500 µl/minute.123 In fact, a 0.5 µl volume of cells has been delivered to the scala media with no significant morphological damage.123 The distribution of stem cells delivered to the organ of Corti, spiral limbus, and spiral ganglion illustrates the widespread migration of the cells from the point of injection.113 It appears that stem cells have the ability to migrate and differentiate over a much wider area of the scala media than was expected. Transplantation and immunological rejection A major consideration for cell therapy is the potential for immunological reactivity when foreign cells are delivered into an organism. Molecular Therapy vol. 16 no. 2 feb. 2008

While these effects are obviously limited by allografts or autologous transplantation, the ethical concerns and practical difficulties associated with the use of human tissue means widespread c­linical application of these forms of transplantation remains unlikely. Therefore, it is pertinent that experimental strategies encompass xenotransplantation, as tissues and cells from organisms such as rats, mice, guinea pigs, and pigs are far more accessible. In fact, significant progress in cell therapy has been facilitated by xenotransplantation, particularly using porcine grafts for liver failure and brain conditions such as Huntington’s disease and epilepsy.125 However, there is always the risk of animal-to-human transmission of new infective agents.125 Several studies have demonstrated successful cross-species transplantation of cells and tissues for treating a number of neurological disorders observed in humans. For example, a study by Gray and colleagues (1999) described the transplantation of multipotent MHP clonal cell lines, derived from the developing hippocampus of a transgenic mouse, into the hippocampus of rats and marmosets with damage to the CA1 cell field, a region affected in Parkinson’s disease.126 The grafted cells survived any immunogenic reaction and remained sufficiently healthy to induce complete recovery of cognitive function.126 It was subsequently shown that the MHP36 cells had migrated to the damaged region and adopted both neuronal and glial phenotypes to reconstitute the CA1 cell field.126 A more promising study by Qu and colleagues (2001) has demonstrated that human stem cells can be successfully grafted into organisms from other species and can even restore function.127 The research group delivered human NSCs into the lateral ventricle of mature (6 month old) and aged (24 month old) rats.127 The aged rats were divided into two groups on the basis of their cognitive function—aged memory impaired and unimpaired. It was found that after human NSC implantation, the majority of aged animals developed cognitive function in the same range as the mature animals and that one aged memory-impaired animal displayed dramatically better behavior than any of the mature animals.127 Further analysis of the transplanted cells with specific markers indicated that they had differentiated into neurons and astrocytes. However, detailed studies comparing the efficacy and safety of allograft, autologous, and xenograft transplantation in the inner ear have not been reported.

Conclusions Recent advances in therapies for hearing loss have resulted in more specific and less traumatic strategies aimed at functional restoration of the auditory system. The introduction of RNAi technology has reduced off-target and nonspecific effects and has provided a potent and relatively safe strategy for targeting specific genes. Examination of stem cell viability and plasticity in the inner ear has provided us with a vehicle for cochlear cell replacement and repair. However, despite this knowledge and improved techniques, no studies have reported regeneration of the auditory system. Significant gaps remain in our knowledge regarding the molecular interactions underpinning auditory function, including the factors required for cellular regeneration and regulation of cochlear gene expression. In addition, practical considerations of stability, duration, trauma, and immunogenicity associated with 233

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therapeutic strategies must be addressed and refined. Until these gaps are filled, the significant potential of these therapeutic strategies for the inner ear will not be realized. Acknowledgments R.J.H.S. is the Sterba Hearing Research Professor, University of Iowa College of Medicine, who supported the project with a National ­Institute on Deafness and Other Communication Disorders grant (RO1 DCO3544) for autosomal dominant nonsyndromic hearing loss. The cell therapy studies at the Murdoch Children’s Research Institute were supported by the ­Garnett Passe and Rodney Williams Memorial Foundation. H.-H.M.D. is a National Health and Medical Research Council Principal Research Fellow. No researchers involved in this study report a conflict of interest. S.D.R. and M.A.B. are employed by Integrated DNA Technologies, (IDT) which offers oligonucleotides for sale similar to some of the compounds described in the manuscript. IDT is however not a publicly traded company and neither author owns any shares or equity in IDT.

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