Environmental inner ear insults often lead to hair cell injury and loss. Therapeutic measures for the prevention of hair cell loss are currently limited. Several reports have demonstrated the applicability of growth factors for hair cell protection. The goal of the experiments presented here was to assess the protective capability of the human GDNF transgene against noise trauma in the guinea pig cochlea. The left ears of guinea pigs were inoculated with a recombinant adenovirus with a human GDNF insert (Ad.GDNF). Four days later, animals were exposed to noise trauma. One week later, animals were sacrificed and hair cells counted in the left (inoculated) and right (non-inoculated) ears. Auditory brainstem thresholds were measured before the inoculation and just prior to sacrifice. Control groups included inoculation with a reporter gene vector (Ad.lacZ) and Ad.GDNF in normal ears with no noise exposure. The results show that intracochlear inoculation with adenovirus into normal ears does not compromise hair cell counts and ABR thresholds. Both Ad.GDNF and Ad.lacZ vectors can protect the cochlear hair cells and hearing from the noise insult. The difference between the protection afforded by Ad.GDNF and that of the Ad.lacZ vector is not statistically significant. The mechanism of Ad.lacZ protection needs to be elucidated. The data demonstrate the general feasibility of gene therapy for over-expression of neurotrophic factors against noise trauma, and emphasize the complexity of the technique and the problems of variability between subjects.
Keywords: guinea pig, cochlea, acoustic trauma, gene therapy, adenovirus, GDNF
|How to cite this article:|
Kawamoto K, Kanzaki S, Yagi M, Stover T, Prieskorn DM, Dolan DF, Miller JM, Raphael Y. Gene-based therapy for inner ear disease. Noise Health 2001;3:37-47
| Introduction|| |
Inner ear disease can result in significant negative impact on the quality of life. The etiology of inner ear disease can be genetic or environmental and lead to hearing and/or balance deficits. The most common environmental inner ear insults, acoustic overstimulation, ototoxic drugs and infections (bacterial or viral) usually target hair cells. Therapeutic measures for the prevention of hair cell loss are limited and interventions for reversing the loss of hair cells are presently unavailable. In some cases hair cells are injured and not lost. In such cases, it is important to develop therapies that may enhance survival and recovery of structure and function in the injured cells. Thus, the three main clinical goals that need to be addressed are prevention, repair and regeneration.
Genes that regulate the ontogeny of the inner ear and its response to insults are rapidly being identified and characterized (Fekete, 1999; Steel and Bussoli, 1999). The gene products of many of these genes have the potential to provide prevention or cure for inner ear trauma.
Delivering protective proteins or other gene products into the inner ear has been shown to afford a significant degree of protection and rescue against acoustic trauma and other insults (Shoji et al., 2000; Staecker et al., 1997; Van De Water et al., 1996). The main disadvantage of this approach is the transient effectiveness of proteins or small therapeutic molecules, due to their degradation. In contrast, if the genes that encode the therapeutic agents can be introduced into the cells and lead to production of the gene product, a more stable type of therapy can be accomplished. Thus, to utilize the genetic information for designing therapies that will prevent or cure inner ear disease, it is necessary to develop gene-based interventions.
Gene-based therapy involves the delivery and expression of a gene, and thereby the availability of the gene product to the cells in the lesion, where it can exert its protective or therapeutic effect (Anderson, 2000; Nabel et al., 1997; Nabel, 1998). If the gene product is a secreted one (i.e. neurotrophic factor), its expression in the vicinity of the target cells may allow it to exert its effect in a paracrine fashion. In many cases, however, the delivered gene must be expressed within the critical cells, necessitating the transfection of a specific cell type. A variety of vectors are available for delivering transgenes into cells. Non-viral vectors are safe but their efficiency is limited and in most cases insufficient for obtaining therapeutic levels of gene expression (Kabanov and Kabanov, 1995; Qin et al., 1998). Viral vectors are currently the most efficient vectors, but each of them has some limiting disadvantages.
Due to the isolated anatomy of the inner ear, this organ is an ideal target for localized gene therapy, without systemic effects. The inner ear is encapsulated in bone, greatly limiting the spread of inoculated material to surrounding tissues. Nevertheless, caution should be exercised in administration of viral vectors because spread of vector to the contralateral ear has been demonstrated for both adeno-associated virus (Lalwani et al., 1998) and adenovirus (Stover et al., 2000a), when applied at relatively high volumes. Thanks to the continuous fluid compartments of the inner ear, vector that is administered in one location (such as a cochleostomy or a trans-round window injection) can be expected to spread throughout the cochlea.
Experiments using adenoviral vectors with a reporter-gene insert demonstrated that in the mature mammalian inner ear, fibroblasts and other cells of connective tissue origin are transduced with high efficiency (Yagi et al., 1999). Hair cells and supporting cells of the normal organ of Corti are not transduced, presently limiting the applicability of adenovirus-based gene therapy in the inner ear to genes encoding secreted and diffusible proteins, such as the neurotrophins.
Neurotrophic factors can influence neuronal development, growth and survival (Shen et al., 1997). Among the known neurotrophic factors are the family of neurotrophins, which includes NGF, BDNF, NT-3 and NT-4/5. Other important neurotrophic factors are IGF-I, IGF-II, TGF-b1, CNTF and the family of GDNF and related molecules. Many of the genes that encode neurotrophic factors (as well as their receptors) have been cloned and sequenced. This knowledge provided insight into the function of each of these genes and their possible clinical use (Shen et al., 1997). It is feasible that therapy based on an over-expression of neurotrophic factors can provide protection against degeneration and enhance repair in the inner ear epithelium.
GDNF, a member of the TGF-β family of growth factor-encoding genes (Lin et al., 1993; Lin et al., 1994), acts via a multi-component receptor complex. GDNFR-α is an extracellular GPI-anchored cell surface coreceptor, also known as α1 (Jing et al., 1996; Jing et al., 1997; Treanor et al., 1996). The transmembrane domain is a tyrosine kinase receptor named RET (Durbec et al., 1996; Taraviras et al., 1999). GDNF and its receptors are expressed in several brain regions (Glazner et al., 1998), in non-neural tissue (Trupp et al., 1995) and in the organ of Corti (Stover et al., 2000b). We have previously demonstrated that inoculation of a viral vector with a human GDNF cassette (designated Ad.GDNF) can protect cochlear cells and hearing from ototoxic trauma (Yagi et al., 1999). We have also shown that viral-mediated overexpression of GDNF can rescue and protect vestibular hair cells from a gentamicin insult (Suzuki et al., 2000). The goal of the experiments presented here was to determine if viral-mediated inner ear expression of the human GDNF transgene can protect auditory hair cells from acoustic trauma.
| Materials and Methods|| |
All animal experiments were approved by the University of Michigan Institutional Committee on the Use and Care of Animals and were performed using accepted veterinary standards. Experimental animals were young pigmented guinea pigs (214-390 gr.) of either sex. Animals were obtained from an outbred colony raised by Murphy's Breeding Laboratory, Inc., Plainfield, IN. The general experimental protocol proceeded according to the following schedule: bilateral auditory brainstem responses (ABRs) at 2, 4, 8 and 20 kHz for establishing hearing baseline, followed by inoculation of the left inner ear with Ad.GDNF or a control solution (day 0), noise exposure (day 4), final ABRs and sacrificing of the animals (day 11). The right (non-inoculated) ears served as controls. The control solution was an adenovirus with a reporter gene insert (Ad.lacZ). The group that received Ad.GDNF followed by noise was designated Ad.GDNF / noise group and had 6 animals. The virus control group was designated Ad.lacZ / noise group and had 8 animals. An additional control group consisted of 4 animals that received Ad.GDNF inoculation with no noise exposure. The initial cohort of animals included 4 additional guinea pigs which were excluded from the report. In three of these animals there was no trauma in the right ears, as determined by both ABRs and cytocochleograms. In the fourth excluded animal the tissue preparation was faulty and precluded a reliable count of hair cells.
To administer the vectors, animals were anesthetized with an intramuscularly administered solution of Rompun (xylazine, 10mg/kg, Bayer, Shawnee Mission, KS) and Ketalar (ketamine HCL, 40mg/kg, Parke Davis, Morris Plains, NJ). Chloramphenicol sodium succinate (30 mg/kg, IM) was administered as prophylaxis, and 0.5 ml of 1% lidocaine HCl was injected subcutaneously for local anaesthesia. The left temporal bone was exposed postauricularly and fenestrated to reveal the basal portion of the cochlea. A cochleostomy was opened as previously described (Stover et al., 1999; Prieskorn and Miller, 2000). Approximately 5 µl of an adenoviral suspension (approximate concentration of 10 10 adenoviral particles per ml in sterile normal Ringer's solution) was slowly injected into the scala tympani perilymph through the cochleostomy. After the inoculation, the cochleostomy was covered with fascia, the bulla was sealed with Durelon and the skin closed with sutures. Subcutaneous postoperative administration of 10 ml of 0.9% saline provided rehydration. Four days after the inoculation, animals were exposed to 4 kHz octave band noise, presented at 115 dB SPL for 5 hours. The generation, amplification and calibration of the noise were as previously described (Yamasoba and Dolan, 1997).
Seven days after the noise exposure, animals were anaesthetized, ABRs were measured, and the animals euthanized. The inner ears were removed and fixed in 4% paraformaldehyde for 2-3 hours. In all animals, the otic capsule, lateral wall, and tectorial membrane were removed, and the bony modiolus with the organ of Corti was carefully detached at the base of the cochlea. At this stage, tissues were permeabilized with 0.3% Triton-X-100 in PBS for 10 minutes, then incubated for 30 minutes with rhodamine phalloidin (Molecular Probes, Eugene, OR) diluted 1:100 in PBS at room temperature. After washing, the turns of the organ of Corti were separated from the modiolus, mounted on microscope slides with Crystal/Mount (Biomeda Co.), and examined for presence or absence of hair cells.
We used an Excel based morphometry program (Yagi et al., 1999) to plot cytocochleograms. In all cases, hair cells were counted in both ears. Statistical significance of differences between inoculated and control (non-inoculated) ears was tested with paired t-tests and signed rank tests. Comparisons between groups were accomplished Kruskal-Wallis ANOVA on ranks. Sigma Stat TM statistical software was used for statistical analysis. Data comparing the inoculated and non-inoculated ears in each group were evaluated using Student's paired t-tests for significance (p<0.05). The threshold shifts across frequencies and the percent of hair cell loss in each ear were also compared among groups using pairwise comparison, with statistical significance adjusted for multiple comparisons (Student -Newman-Keuls).
| Results|| |
A daily postoperative inspection revealed a complete recovery of the animals, with no head tilt, loss of appetite, or any other signs of earspecific or systemic toxicity in any of the guinea pigs. Ad.GDNF inoculation into the left ear of normal guinea pigs (without noise exposure) did not cause a significant loss of hair cells [Figure - 1]. Auditory thresholds remained unchanged [Figure - 2].
The extent of lesion that was generated by the noise exposure was demonstrated by assessment of the morphology of the right ears [Figure - 3]A and C. The structural lesion affected mostly the outer hair cell population. It was accompanied by a threshold shift of 35- 50 dB, depending on frequency [Figure - 4].
Left ears in the Ad.GDNF group were clearly protected from the acoustic trauma. In some areas, the population of hair cells was intact [Figure - 3]B and D. The protection was statistically significant (p<0.05) when left ears were compared to the right (untreated) ears [Figure - 5]. The extent of hair cell protection afforded by the Ad.lacZ inoculation [Figure - 6] was also significant (p<0.05). There was no statistically significant difference between the degree of hair cell protection afforded by the Ad.lacZ vector compared to that of Ad.GDNF. Threshold protection by Ad.lacZ was also noted, but the level of protection was not statistically significant [Figure - 7].
Taken together, the data indicate that intracochlear inoculation with adenovirus into normal ears does not compromise hair cell counts and ABR thresholds. The results also demonstrate that both Ad.GDNF and Ad.lacZ vectors can protect the cochlea from a noise insult.
| Discussion|| |
The data demonstrate four major points. First, in all animals (except one) in the Ad.GDNF / noise group, the hearing thresholds and the preservation of hair cells were better in the left (Ad.GDNF-inoculated) ears than the right (noninoculated) ears. Second, some of the left ears in the Ad.lacZ group were also robustly protected as compared to the right (contralateral) ears. Third, Ad.GDNF by itself is not toxic to the ear. Finally, the variability in the data was pronounced.
The feasibility of using growth factors for protection and rescue in the inner ear has been established in several experimental models (Shoji et al., 2000; Staecker et al., 1997; Van De Water et al., 1996; Ylikoski et al., 1998). The possibility to accomplish such protective effects by over-expression of growth factors via gene transfer has also been demonstrated, in the auditory (Yagi et al., 1999) and vestibular epithelium (Suzuki et al., 2000). Protective effects on auditory neurons have also been demonstrated (Ernfors et al., 1996; Miller et al, 1997; Van De Water et al., 1996). The data presented here extend these observations to protection against noise trauma.
The most intriguing result is the protection afforded by the Ad.lacZ vector. Protection by Ad.lacZ has been previously observed in experiments with ototoxic drugs (Yagi et al., 1999). However, the extent of LacZ protection is greater in the noise experiments reported here than in the previous studies using ototoxic insults. The mechanism of protection afforded by the Ad.lacZ against acoustic trauma is unclear. We speculate that the mechanism of protection of Ad.lacZ is based on a stressresponse cascade. One possibility is that the presence of the viral vector in the inner ear leads to cytokine expression and signaling, causing a mild immune response, which, in turn, increases the resistance of the tissue to trauma. There is evidence from other biological systems that cytokines can play protective roles by enhancing secretion of neurotrophins and other growth factors (Appel et al., 1997; Ebadi et al., 1997; Lindsay and Yancopoulos, 1996).
The lack of toxicity of Ad.GDNF in normal (non-traumatized) ears is of importance to the on-going efforts to pursue inner ear gene therapy technology. These data demonstrate that in experimental animals, administration of a small amount of fluid into the perilymph, the presence of a viral vector in the inner ear, and the (presumed) over-expression of GDNF are all compatible with preservation of normal thresholds and hair cell counts. It is, therefore, likely that along with the improvement of gene transfer vectors and the identification of genes with therapeutic potential, the feasibility for clinically applicable inner ear gene therapy will increase.
The mechanism of action of GDNF in protecting the inner ear against acoustic trauma is unclear. The present study was not designed to address this issue. If anything, the protective effects of Ad.lacZ confound our ability to explain the protective effect of GDNF. Based on work with cultured cells and on data from other systems, it has been determined that GDNF can prevent cell death by scavenging oxygen free radicals, upregulating anti-apoptotic genes, or blocking Ltype calcium channels (Springer and Kitzman, 1998). Further work in the organ of Corti is necessary to identify the protective pathways activated by exogenous GDNF in this organ.
While the ability of gene therapy using secreted gene products to provide protection and rescue in the inner ear has been clearly demonstrated (Suzuki et al., 2000; Yagi et al., 1999), the variability in the data is higher than desirable. We are currently attempting to determine which of the parameters contributes to the variability, and how it could be reduced. Some problems are clearly associated with the delivery of vectors. It is expected that future vectors will have minimal toxicity, allow for regulated timing of cellspecific gene expression and induce no immunogenicity. Progress towards achieving these goals is being made (Amalfitano, 1999; Amalfitano et al., 1996; Amalfitano et al., 1998; Hartigan-O'Connor et al., 1999).
The data demonstrate the complexity and technical difficulties involved in the delivery of transgenes into the inner ear and, in parallel, the optimistic potential for such treatment as it improves with time. Rapid progress in vector technology is likely to yield vectors that are nontoxic and non-immunogenic. Ideal vectors will infect a selective population of cells with high efficiency, and have an inducible promoter allowing regulated expression of the inserted gene. Gutted adenovirus technology (Amalfitano, 1999; Hartigan-O'Connor et al., 1999) and the rapid development of non-viral vectors are likely to make gene therapy for environmental and genetic inner ear disease a reality.
In conclusion, the data we present demonstrate the feasibility of gene therapy for overexpression of neurotrophic factors for preventive and therapeutic application in the inner ear. The data also point to the complexity of the technical aspects of inner ear gene transfer, which need to be resolved before these procedures become clinically feasible.
| Acknowledgements|| |
We thank Lisa Beyer and Gary Dootz for excellent technical assistance. Support was provided by NIH NIDCD grants P01 DC00078 (DFD and YR), R01 DC01634 (YR) and R01 DC04058 (JMM).
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Kresge Hearing Research Institute, The University of Michigan Medical School, Rm. 9303, MSRB 3, Ann Arbor, MI 48109-0648
Source of Support: None, Conflict of Interest: None
[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6], [Figure - 7]