Retinitis pigmentosa type 45 (RP45) is an autosomal-recessively inherited blinding disease caused by mutations in the cyclic nucleotide-gated channel subunit beta 1 (CNGB1) gene. In this study, we developed and tested a novel gene supplementation therapy suitable for clinical translation. To this end, we designed a recombinant adeno-associated virus (rAAV) vector carrying a genome that features a novel human rhodopsin promoter (hRHO194) driving rod-specific expression of full-length human CNGB1 (rAAV5.hCNGB1). rAAV5.hCNGB1 was evaluated for efficacy in the Cngb1 knockout (Cngb1^−/−) mouse model of RP45. In particular, increasing doses of rAAV5.hCNGB1 were delivered through single subretinal injection in 4-week-old Cngb1^−/− mice and the treatment effect was assessed over a follow-up period of 9 months at the level of (1) retinal morphology, (2) retinal function, (3) vision-guided behavior, and (4) transgene expression. We found that subretinal treatment with rAAV5.hCNGB1 resulted in efficient expression of the human CNGB1 protein in mouse rods and was able to normalize the expression of the endogenous mouse CNGA1 subunit, which together with CNGB1 forms the native heterotetrameric cyclic guanosine monophosphate-gated cation channel in rod photoreceptors. The treatment led to a dose-dependent recovery of rod photoreceptor-driven function and preservation of retinal morphology in Cngb1^−/− mice. In summary, these results demonstrate the efficacy of hCNGB1 gene supplementation therapy in the Cngb1^−/− mouse model of RP45 and support the translation of this approach toward future clinical application.

中文翻译：

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视网膜下 rAAV5.hCNGB1 基因治疗在色素性视网膜炎 Cngb1 基因敲除小鼠模型中的体内效力测试

45 型色素性视网膜炎 (RP45) 是一种由环状核苷酸门控通道亚基 β 1 ( CNGB1 ) 基因突变引起的常染色体隐性遗传致盲疾病。在这项研究中，我们开发并测试了一种适用于临床转化的新型基因补充疗法。为此，我们设计了一种重组腺相关病毒 (rAAV) 载体，该载体携带的基因组具有新的人类视紫红质启动子 (hRHO194)，可驱动全长人类CNGB1 (rAAV5.hCNGB1) 的杆状特异性表达。评估了 rAAV5.hCNGB1 在 RP45 的Cngb1敲除 ( Cngb1 ^-/- ) 小鼠模型中的功效。特别是，增加剂量的 rAAV5.hCNGB1 是通过单次视网膜下注射在 4 周大的Cngb1 ^-/-小鼠和治疗效果在 9 个月的随访期内在 (1) 视网膜形态、(2) 视网膜功能、(3) 视觉引导行为和 (4) 转基因表达水平进行评估. 我们发现用 rAAV5.hCNGB1 进行视网膜下治疗导致人 CNGB1 蛋白在小鼠视杆细胞中的有效表达，并且能够使内源性小鼠 CNGA1 亚基的表达正常化，它与 CNGB1 一起形成天然异四聚体环状单磷酸鸟苷门控阳离子通道在杆状感光器中。该治疗导致Cngb1 ^-/-小鼠的视杆细胞驱动功能和视网膜形态的剂量依赖性恢复。总之，这些结果证明了hCNGB1的功效RP45 的Cngb1 ^-/-小鼠模型中的基因补充疗法，并支持将这种方法转化为未来的临床应用。