PDF(Pubmed)
Abstract：
Congenital stationary night blindness (CSNB) is a group of inherited retinal diseases in which either rod-to-ON-bipolar cell (ON-BC) signaling, or rod function is affected leading to impaired vision under low light conditions. One type of CSNB is associated with defects in genes (NYX, GRM6, TRPM1, GPR179, and LRIT3) involved in the mGluR6 signaling cascade at the ON-BC dendritic tips. We have previously characterized a canine model of LRIT3-CSNB and demonstrated short-term safety and efficacy of an ON-BC targeting AAV-LRIT3 (AAVK9#4-shGRM6-cLRIT3-WPRE) gene therapy. Herein, we demonstrate long-term functional recovery and molecular restoration following subretinal injection of the ON-BC targeting AAV-LRIT3 vector in all eight treated eyes for up to 32 months. Following subretinal administration of the therapeutic vector, expression of the LRIT3 transgene, as well as restoration of mGluR6 signaling cascade member TRPM1, were confirmed in the outer plexiform layer (OPL) of the treated area. However, further investigation of the transgene LRIT3 transcript expression by RNA in situ hybridization (RNA-ISH) revealed off-target expression in non-BCs including the photoreceptors, inner nuclear, and ganglion cell layers, despite the use of a mutant AAVK9#4 capsid and an improved mGluR6 promoter designed to specifically transduce and promote expression in ON-BCs. While the long-term therapeutic potential of AAVK9#4-shGRM6-cLRIT3-WPRE is promising, we highlight the necessity for further optimization of AAV-LRIT3 therapy in the canine CSNB model prior to its clinical application.
摘要：
先天性静止性夜盲症（CSNB）是一组遗传性视网膜疾病，其中杆-ON-双极细胞（ON-BC）信号传导，或杆功能受到影响，导致弱光条件下的视力受损。一种类型的CSNB与基因缺陷有关(NYX，GRM6，TRPM1，GPR179和LRIT3）参与ON-BC树突状尖端的mGluR6信号级联。我们先前已经表征了LRIT3-CSNB的犬模型，并证明了靶向AAV-LRIT3（AAVK9＃4-shGRM6-cLRIT3-WPRE）基因治疗的ON-BC的短期安全性和有效性。在这里，我们证明了在所有8只接受治疗的眼睛中视网膜下注射针对ON-BC的AAV-LRIT3载体长达32个月后，长期功能恢复和分子恢复。在视网膜下施用治疗载体后，LRIT3转基因的表达，以及mGluR6信号传导级联成员TRPM1的恢复，在治疗区域的外部丛状层（OPL）中得到证实。然而,通过RNA原位杂交（RNA-ISH）对转基因LRIT3转录本表达的进一步研究揭示了在包括光感受器在内的非BCs中的脱靶表达，内核，和神经节细胞层，尽管使用了突变体AAVK9#4衣壳和设计用于特异性转导和促进ON-BC中表达的改进的mGluR6启动子。虽然AAVK9#4-shGRM6-cLRIT3-WPRE的长期治疗潜力是有希望的，我们强调了在临床应用前进一步优化犬CSNB模型中AAV-LRIT3治疗的必要性.
