Wilkes Rebecca P, Ward Dan A, Newkirk Kim M, Adams Joleen K, Kania Stephen A
Department of Biomedical and Diagnostic Research, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA.
Am J Vet Res. 2013 Feb;74(2):243-7. doi: 10.2460/ajvr.74.2.243.
To evaluate agents used for delivery of small interfering RNAs (siRNAs) into feline corneal cells, toxicity of the delivery agents, and functionality of anti-feline herpesvirus 1 (FHV-1)-specific siRNA combinations.
Feline primary corneal cells and 19 six-month-old colony-bred cats.
siRNA delivery into corneal cells via various delivery agents was evaluated via flow cytometric detection of labeled siRNAs. Cellular toxicity was evaluated with a proliferation assay. Functionality was tested via quantitative reverse transcriptase PCR assay, plaque assay, and flow cytometry. In vivo safety was evaluated with an ocular scoring method following topical application of delivery agents containing siRNAs into eyes. Corneal biopsy specimens were used to assess safety and uptake of siRNAs into corneal cells.
Use of 3 delivery agents resulted in > 95% transfection of primary corneal cells. Use of a peptide for ocular delivery yielded approximately 82% transfection of cells in vitro. In cultured corneal cells, use of the siRNA combinations resulted in approximately 76% to 89% reduction in FHV-1-specific mRNA, 63% to 67% reduction of FHV-1-specific proteins in treated cells, and 97% to 98% reduction in FHV-1 replication. The agents were nonirritating in eyes, caused no substantial clinical ocular signs, and were nontoxic. Histologically, corneal epithelium and stroma were normal in treated cats. However, none of the agents were effective in delivering siRNAs into the corneal cells in vivo.
The tested anti-FHV-1-specific siRNAs could potentially be used as a treatment for FHV-1 if a successful means of in vivo delivery can be achieved.
评估用于将小干扰RNA（siRNA）递送至猫角膜细胞的试剂、递送试剂的毒性以及抗猫疱疹病毒1型（FHV-1）特异性siRNA组合的功能。
猫原代角膜细胞和19只6月龄群体饲养的猫。
通过流式细胞术检测标记的siRNA，评估经由各种递送试剂将siRNA递送至角膜细胞的情况。通过增殖试验评估细胞毒性。通过定量逆转录聚合酶链反应试验、蚀斑试验和流式细胞术测试功能。在将含有siRNA的递送试剂局部应用于眼睛后，采用眼部评分方法评估体内安全性。使用角膜活检标本评估siRNA进入角膜细胞的安全性和摄取情况。
使用3种递送试剂可使原代角膜细胞的转染率>95%。使用一种用于眼部递送的肽在体外可使细胞的转染率约为82%。在培养的角膜细胞中，使用siRNA组合可使FHV-1特异性mRNA减少约76%至89%，使处理细胞中FHV-1特异性蛋白减少63%至67%，并使FHV-1复制减少97%至98%。这些试剂对眼睛无刺激性，未引起明显的临床眼部体征，且无毒。组织学检查显示，治疗猫的角膜上皮和基质正常。然而，没有一种试剂在体内将siRNA有效递送至角膜细胞。
如果能够实现成功的体内递送方法，所测试的抗FHV-1特异性siRNA有可能用于治疗FHV-1。
