The effect of whirlin isoforms on the efficacy of inner ear gene therapy in whirlin mutant mice

Claire Cheng, Kevin Isgrig, Jiang Liang Zhu, Wade ChienNational Institute of Deafness and Communication Disorders, National Institutes of Health, Bethesda, MD

Introduction

Animal Surgery: P0-P5 pups were anesthetized, and a postauricular incision was made, exposing the neck muscles. The posterior canal was identified. As seen in Figure 1, a glass micropipette was loaded with 1 μl of AAV8-whirlin (1x1013 genome copies per ml) and injected into the posterior canal. The long isoform of whirlin cDNA was used.

Methods

Chien, W. W., Isgrig, K., Roy, S., Belyantseva, I. A., Drummond, M. C., May, L. A., … Cunningham, L. L. (2016). Gene Therapy Restores Hair Cell Stereocilia Morphology in Inner Ears of Deaf Whirler Mice. Molecular Therapy, 24(1), 17–25. http://doi.org/10.1038/mt.2015.150

Results

Several studies have shown that AAV8-whirlin injections improved hearing and balance functions in whirler mice, but did not restore full hearing recovery. 1,2 Two possible reasons for insufficiency is the limited OHC infection rate observed and the use of only the long isoform of whirlin cDNA. In this study, the Anc80 virus was used to examine its effects on hearing results on whirler mice in comparison to the AAV8 virus. Our data show the Anc80 virus has a much higher infection efficiency in both IHCs and OHCs compared to AAV8 virus. We also showed the Anc80 virus did not result in improved hearing despite that it did restore stereocilia length. In addition, we suggest the short isoform of whirlin cDNA did not improve hearing results. We plan to look for other aspects of gene therapy that can restore full hearing recovery in mouse models of hearing loss.

Discussion

• Anc80 virus had a higher infection rate on cochlear outer hair cells than AAV8 virus

• Addition of short isoform does not improve hearing results in Whrntm1b Hom mice

Conclusions

Mice. Molecular Therapy, 24(1), 17–25. http://doi.org/10.1038/mt.2015.150

References and Acknowledgements

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2. Isgrig, K., Shteamer, J. W., Belyantseva, I. A., Drummond, M. C., Fitzgerald, T. S., Vijayakumar, S., … Chien, W. W. (2017). Gene Therapy Restores Balance and Auditory Functions in a Mouse Model of Usher Syndrome. Molecular Therapy, 25(3), 780–791. http://doi.org/10.1016/j.ymthe.2017.01.0073. Ebrahim, S., Ingham, N. J., Lewis, M. A., Rogers, M. J. C., Cui, R., Kachar, B., … Steel, K. P. (2016). Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia. Cell Reports, 15(5), 935–943. http://doi.org/10.1016/j.celrep.2016.03.081

4. Pan, B., Askew, C., Galvin, A., Heman-Ackah, S., Asai, Y., Indzhykulian, A. A., … Géléoc, G. G. S. (2017). Gene Therapy Restores Auditory and Vestibular Function in a Mouse Model of Usher Syndrome Type 1c. Nature Biotechnology, 35(3), 264–272. http://doi.org/10.1038/nbt.3801

Anc80 AAV infects both IHCs and OHCs with high efficiencyPrevious studies have explored the effectiveness of gene therapy in restoring the hair cell stereocilia architecture in whirler mice, which is deaf due to dysmorphic, short stereocilia. 1,2 They showed that AAV8-whirlin injections improved hearing and balance functions in whirler mice, but did not restore full hearing recovery. One possible reason for this aspect is that the limited outer hair cell infection rate observed is insufficient for hearing recovery. 4 Another possible reason is that the studies utilized the long isoform of whirlin cDNA, whereas the short whirlin isoform may also play an important role in establishing full recovery. 1 In this study, we assess the effect of AAV8 and Anc80 serotypes as well as the a short isoform of whirlin on improving the hearing of whirlin mutant mice.

Increased OHC infection with Anc80-whirlin did not result in improved hearing.

Addition of short isoform did not improve hearing results

Anc80-whirlin resulted in restoration of stereocilia length in whirler mice

Animal Surgery: P0-P5 pups were anesthetized, and a postauricular incision was made, exposing the neck muscles. The posterior canal was identified. A glass micropipette was loaded with 1 μl of AAV8-whirlin or with 1 μl of Anc80- whirlin (1x1013 genome copies per ml) and injected into the posterior canal. The long isoform of whirlin cDNA was used.

Auditory Brainstem Response (ABR): ABR testing was used to evaluate hearing sensitivity of whirler mice between 1-3 months of age. ABR was recorded using the Tucker Davis Technologies system (RZ6 Multi I/O Processor, Tucker-Davies Technologies). ABR thresholds were measured at 4, 8, 16, and 32 kHz, and 512-1024 responses were averaged at each stimulus level. Thresholds were determined by visual inspection of the waveforms and were defined at the lowest stimulus level at which any wave could be reliably detected.

Immunohistochemistry: Whole mounts of the cochlea were obtained and dissected into base, middle, and apical turns. Antibodies against myosin VIIA and whirlin as well as Rhodamine-phalloidin were applied. Z-stacks were created using a Zeiss LSM780 confocal microscope. Volocity 3D image analysis software was used to measure stereocilia length in infected and non-infected hair cells. The samples were dismounted and processed for SEM as previously described.

1. Chien, W. W., Isgrig, K., Roy, S., Belyantseva, I. A., Drummond, M. C., May, L. A., … Cunningham, L. L. (2016). Gene Therapy Restores Hair Cell Stereocilia Morphology in Inner Ears of Deaf Whirler Mice. Molecular Therapy, 24(1), 17–25. http://doi.org/10.1038/mt.2015.150

Figure 1: Infected IHCs and OHCs showed GFP expression (green) that colabeled with myosin 7a (red). The Anc80 infected IHCs and OHCs showed substantial amount of GFP expression while AAV8 infected IHCs showed minimal GFP expression (arrowheads). Quantification of IHC and OHC show a significant difference between AAV8 and Anc80 infection rates.

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Figure 2: (A) The IHC in whirler mutant (Whrn wi/wi) with Anc80 infection had restored stereocilia length and close to 100% hair cell infection rate. (B) In contrast, the IHC in whirler mutant (Whrn wi/wi) with no Anc80 infection had short stereocilia length and no hair cell infection.

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Figure 3: One month after Anc80-whirlin gene therapy, ABR threshold was detected at around 95 dB SPL in a whirler mouse. This is not statistically significant from a whirler mouse gone through AAV8 gene therapy, which had an ABR threshold at 100 dB SPL.

Figure 4: Whrntm1b was used to determine whether the addition of a short isoform of cDNA would improve hearing results in whirler mutants (Whrn wi/wi). Both ABR thresholds for the whrntm1b Hom w/ AAV8 whirlin and whrntm1b Hom were detected at around 90 dB SPL, showing no improvement in hearing results using the short isoform.

Whrntm1b Hom w/ AAv8 whirlin infection