Understanding The Visual And Auditory Defect In Ush2a Mouse Model



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Usher syndrome (USH) is the most common form of dual deafness and irreversible vision loss found in patients worldwide. USH2 is the most prevalently occurring sub type, accounting for ~50 to 75% of USH clinical cases. Patients with USH2 suffer from congenital hearing loss and progressive vision loss beginning from adolescence. Mutations in USH2A (usherin) account for ~80% of USH2 patients; making it the most common genetically mutated gene among USH patients. Usherin has been detected in the photoreceptors of the retina and in the developing inner ear cell stereocilia. The USH2A gene is very large and attempting gene therapy with conventional viral delivery is not easily attainable due to limited payload capacity of commonly used Adeno-associated viruses (AAV) ( <4.7 kbp.) To understand the mechanism underlying hearing and visual impairments, a knock in (KI) mouse model (Ush2adelG/delG) was developed using one of the most prevalent human mutations of USH2A, 2299delG. While other models for Ush2a exist, no other KI model has been able to successfully mimic the genetic mutations found in humans. This model will be valuable to investigate possible delivery methods for USH2A. Retinal phenotype in the KI model was found to follow similar progression as patients, with a notable gradual loss of vison that became apparent at older ages, along with concomitant photoreceptor degeneration. More in-depth analysis found that this was due to the mislocalization of the KI mutant protein and a subsequent mislocalization of its other USH2 interacting partners. Along with patients exhibiting phenotypes associated with homozygous mutations of USH2A, there is also a prevalence of cases involving heterozygous mutations of USH2A in combination with mutations in other USH or non-USH genes that results in varying degrees of phenotype. Backcrossing of Ush2adelG/delG with three knockouts of photoreceptor-specific proteins, rhodopsin (Rho-/-), ABCA4-/-, and ROM1-/-, resulted in double heterozygous of digenic mutants with a mixture of retinal phenotype. The combination of heterozygous USH2A with rhodopsin (Rho+/-/Ush2adelG/+) led to a surprising protective effect of retinal phenotype. We next evaluated the cochlear phenotype of the KI model, showing congenital hearing loss in the lower frequency range, particularly at 8 and 11 kHz, in the auditory brainstem response (ABR) tone tests. Associated stereocilia of the inner hair cells in the apical portion of the cochlea were correspondingly found to be disorganized. Like the retina, the Ush2adelG/delG mutant protein was found to be mislocalized while the associated proteins were found to be properly localized to the stereocilia of the hair cells. Following the analysis and verification of the KI model as a viable model for therapeutic testing, the next step was to develop a therapeutic approach for both the retina and cochlea. With one major challenge of therapy for USH2 being its very large gene, development of an alternative, non-viral delivery, method is needed and an example of such an approach is by encapsulating the therapeutic gene using hyaluronic acid nanospheres (NSs). Delivery approaches for the eye, specifically targeting the retina, using NSs in conjunction with a small molecule, sulfotyrosine, have shown great promise. Preliminary studies using NSs filled with plasmid DNA with GFP expression cassette under the control of ubiquitous promotion (chicken beta actin with CMV enhance, CAG) led to noticeable GFP expression in retinal cells following intravitreal injections. No GFP was detected with the plasmid DNA alone. Along with the retina, delivery to the inner ear cells of the cochlea was also a goal. Preliminary injections of the NSs in P0-P1 round window injections into the cochlea showed no functional differences between the injected and non-injected (contralateral) ears, indicating no major toxic effect caused by the NSs. Future work will look more in depth at the possibility of delivering native USH2A genes into the retina and cochlea as a potential therapeutic options.



Usher Syndrome, Retina, Cochlea, Usherin, Ush2a


Portions of this document appear in: Crane, Ryan, Shannon M. Conley, Muayyad R. Al-Ubaidi, and Muna I. Naash. "Gene therapy to the retina and the cochlea." Frontiers in neuroscience 15 (2021): 652215; and in: Eblimit, A., M.S. Makia, D. Strayve, R. Crane, S.M. Conley, T. Sinha, G. Acharya, M.R. Al-Ubaidi, and M.I. Naash, Co-Injection of Sulfotyrosine Facilitates Retinal Uptake of Hyaluronic Acid Nanospheres Following Intravitreal Injection. Pharmaceutics, 2021.