Impact of Vital Dyes on Cell Viability and Transduction Efficiency of AAV Vectors Used in Retinal Gene Therapy Surgery: An In Vitro and In Vivo Analysis.
Impact of Vital Dyes on Cell Viability and Transduction Efficiency of AAV Vectors Used in Retinal Gene Therapy Surgery: An In Vitro and In Vivo Analysis.
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DOI：
10.1167/tvst.6.4.4
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发表时间：
2017-07
影响因子：
3
通讯作者：
MacLaren RE
中科院分区：
文献类型：
--
作者：
Salvetti AP;Patrício MI;Barnard AR;Orlans HO;Hickey DG;MacLaren RE
使用腺相关病毒（AAV）载体通过视网膜下注射治疗遗传性视网膜变性。在后期，正常解剖结构的改变可能导致视网膜切开术、视网膜脱离延伸和矢量扩散的视觉困难。重要的染料可能是有用的手术辅助，但其安全性和对AAV转导的影响在很大程度上是未知的。采用aav2 -绿色荧光蛋白（GFP）报告基因检测不同感染倍数下荧光素钠（SF）、膜蓝（MB）和膜蓝双组分（DB）对体外人胚胎肾细胞的影响。流式细胞术分析细胞活力和转导效率。测定对定量(q)PCR滴度的影响。将平衡盐溶液（BSS）或稀释DB （BSS比1:5）在视网膜下注入C57BL/6J小鼠左右眼（n = 12）。采用光学相干断层扫描、自体荧光、适应暗、适应光的全视场视网膜电图分析视网膜结构和功能。DB和MB在任何浓度下均无毒，SF仅在未稀释时无毒。染料的存在对基因组滴度没有不利影响。由于配方中存在表面活性剂，DB甚至增加了该值。染料不影响AAV2-GFP的转导效率。视网膜下给药后未见结构和功能毒性作用。只有未稀释的SF影响细胞活力。对qPCR滴度和转导效率无影响。在视网膜下给药时，DB没有毒性，并提高滴度准确性。因此，在基因治疗手术中，DB可能是一种安全有益的辅助手段。这篇论文可能会引起视网膜基因治疗界的兴趣：这是一篇“从实验室到床边”的研究论文，关于在基因治疗手术中使用染料作为手术辅助的潜在用途。我们已经在体外和体内模型中测试了潜在的毒性和对转导效率的影响。
Treatment of inherited retinal degenerations using adeno-associated viral (AAV) vectors involves delivery by subretinal injection. In the latter stages, alteration of normal anatomy may cause difficulty in visualizing the retinotomy, retinal detachment extension, and vector diffusion. Vital dyes may be useful surgical adjuncts, but their safety and impact on AAV transduction are largely unknown. The effects of Sodium Fluorescein (SF), Membrane Blue (MB), and Membrane Blue Dual (DB) at a range of dilutions were assessed on human embryonic kidney cells in vitro using an AAV2-green fluorescent protein (GFP) reporter at different multiplicities of infection. Flow cytometry analysis was performed to assess both cell viability and transduction efficiency. The effect on quantitative (q)PCR titer was determined. Balanced salt solution (BSS) or dilute DB (1:5 in BSS) were delivered subretinally into left/right eyes of C57BL/6J mice (n = 12). Retinal structure and function were analyzed by optical coherence tomography, autofluorescence, dark-and light-adapted full-field electroretinography. DB and MB were not toxic at any concentration tested, SF only when undiluted. The presence of dyes did not adversely affect the genomic titer. DB even increased the values, due to presence of surfactant in the formulation. AAV2-GFP transduction efficiency was not reduced by the dyes. No structural and functional toxic effects were observed following subretinal delivery of DB. Only undiluted SF affected cell viability. No effects on qPCR titer and transduction efficiency were observed. DB does not appear toxic when delivered subretinally and improves titer accuracy. DB may therefore be a safe and helpful adjunct during gene therapy surgery. This paper might be of interest to the retinal gene therapy community: it is a “bench to bedside” research paper about the potential use of dyes as a surgical adjunct during the gene therapy surgery. We have tested the potential toxicity and impact on transduction efficiency in an in vitro and in vivo model.
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影响因子：
13.7
作者：
Jackson, Timothy L.;Kwan, Anthony S. L.;Aylward, William
通讯作者：
Aylward, William
影响因子：
4.4
作者：
Morales, Maria-Celia;Freire, Vanesa;Corcostegui, Gonzalo
通讯作者：
Corcostegui, Gonzalo
影响因子：
1.6
作者：
Khanduja, Sumeet;Sinha, Subijay;Vohra, Rajpal
通讯作者：
Vohra, Rajpal
影响因子：
5.4
作者：
Zolotukhin, S;Potter, M;Muzyczka, N
通讯作者：
Muzyczka, N
影响因子：
12.4
作者：
Bennicelli, Jeannette;Wright, John Fraser;Bennett, Jean
通讯作者：
Bennett, Jean