Abstract
Purpose :
Retinitis pigmentosa resulting from rhodopsin mutations necessitates the suppression of mutant rhodopsin, coupled with precise control over rhodopsin replacement. Excessive rhodopsin expression proves detrimental to retinal health. This research aims to determine the optimal mirtron vector design and dosage to achieve a harmonious in vivo balance between rhodopsin knockdown and replacement.
Methods :
In Rho+/-, hP23H+ mice, subretinal injections were administered at 3-4 weeks of age. AAV8 vectors carrying RHOp.2xM.coRHO (double mirtron) or RHOp.3xM.coRHO (triple mirtron) were delivered at doses of 1E+09 (high dose), 5E+08 (medium dose), or 2E+08 (low dose) genome copies per eye. At 4 weeks post-injection, eyes were taken optical coherence tomography assessment and RNA extraction. Detection of human P23H and replacement rhodopsin was accomplished through TaqMan assay.
Results :
Retinal thickness measurements in AAV-injected cohorts did not exhibit a significant reduction compared to sham-injected eyes. However, whole retina thickness measurements in the superior retina showed a significant reduction in comparison to uninjected eyes across the sham, double mirtron medium dose, low dose, and triple mirtron high dose cohorts. Measurements of the inferior retina demonstrated less variability, with only the double mirtron medium dose cohort significantly reduced compared to uninjected eyes. Notably, the inferior photoreceptor layer exhibited a significant increase in thickness in the triple mirtron high dose and medium dose cohorts. Transcript analysis revealed a substantial knockdown of P23H compared to sham-injected eyes for the double mirtron high dose, triple mirtron high dose, and medium dose cohorts. Furthermore, codon-modified rhodopsin was detected in all AAV treatment groups, with supplementation ratios relative to P23H in sham-injected eyes approximately 5:1 in double and triple mirtron high dose groups, and medium and low dose ratios ≤1:1.
Conclusions :
In vivo, optimal knockdown of human P23H rhodopsin transcripts was achieved, with concurrent attainment of optimal rhodopsin replacement levels at high and medium doses. Enhancements in inferior retinal thickness were noted in eyes administered the triple mirtron vector at high and medium doses, indicating the prospect of utilizing artificial mirtrons in RNA replacement for retinal gene therapy.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.