Characterization of Rcbtb1 Knockout Mice and Evaluation of AAV2-RCBTB1 Gene Replacement Therapy.

Purpose: Biallelic pathogenic variants in the RCC1 and BTB domain-containing protein 1 (RCBTB1) gene cause an adult-onset retinal dystrophy. Here, we generated a knockout mouse model of RCBTB1 deficiency for the evaluation of RCBTB1 gene therapy. Methods: Rcbtb1-knockout (KO) mice were generated with a homozygous deletion removing exons 2 and 3 of Rcbtb1. Wild-type (WT) and Rcbtb1-KO mice were assessed by optical coherence tomography and electroretinography at 3, 8, and 14 months of age. Retinal ultrastructure was assessed by transmission electron microscopy. Subretinal injections of adeno-associated virus 2 (AAV2)-RCBTB1 or AAV2-enhanced green fluorescent protein (EGFP) vector were performed at 2 months, and mice were analyzed at 8 months. Retinal gene expression was assessed by quantitative PCR and immunohistochemistry. Results: Retinal Rcbtb1 expression was absent in Rcbtb1-KO mice. Eight-month-old Rcbtb1-KO mice showed reduced outer retinal thickness compared with WT mice. Ultrastructural analysis demonstrated increased mitochondrial damage in retinal pigment epithelial cells and increased frequencies of mitochondria with oxidative inclusions in photoreceptor inner segments in 8-month-old Rcbtb1-KO mice. Degenerating retinal pigment epithelium (RPE) and photoreceptors were observed in Rcbtb1-KO mice. Bruch's membrane appeared thicker in Rcbtb1-KO mice and contained druse-like deposits. Treatment with AAV2-RCBTB1 induced sustained RCBTB1 expression and preserved outer retinal thickness in 8-month-old Rcbtb1-KO mice. Conclusions: Rcbtb1-KO mice showed accelerated outer retinal thinning, increased mitochondrial damage in the RPE, and photoreceptor apoptosis. AAV2-RCBTB1 vectors induced long-term retinal expression of RCBTB1 and prevented retinal thinning in Rcbtb1-KO mice. Translational Relevance: Rcbtb1-KO mice provide a useful animal model for modeling RCBTB1 deficiency and preclinical screening of novel treatments.