Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Center for Nanomedicine, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Curr Protoc. 2022 Dec;2(12):e607. doi: 10.1002/cpz1.607.
The advent of the first-ever retinal gene therapy product, involving subretinal administration of a virus-based gene delivery platform, has garnered hope that this state-of-the-art therapeutic modality may benefit a broad spectrum of patients with diverse retinal disorders. On the other hand, clinical studies have revealed limitations of the applied delivery strategy that may restrict its universal use. To this end, intravitreal administration of synthetic gene-delivery platforms, such as polymer-based nanoparticles (PNPs), has emerged as an attractive alternative to the current mainstay. To achieve success, however, it is imperative that synthetic platforms overcome key biological barriers in human eyes encountered following intravitreal administration, including the vitreous gel and inner limiting membrane (ILM). Here, we introduce a series of experiments, from the fabrication of PNPs to a comprehensive evaluation in relevant experimental models, to determine whether PNPs overcome these barriers and efficiently deliver therapeutic gene payloads to retinal cells. We conclude the article by discussing a few important considerations for successful implementation of the strategy. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation and characterization of PNPs Basic Protocol 2: Evaluation of in vitro transfection efficacy Basic Protocol 3: Evaluation of PNP diffusion in vitreous gel Basic Protocol 4: Ex vivo assessment of PNP penetration within vitreoretinal explant culture Basic Protocol 5: Assessment of in vivo transgene expression mediated by intravitreally administered PNPs.
首款视网膜基因治疗产品的问世，涉及基于病毒的基因传递平台的视网膜下给药，为这一最先进的治疗方式可能使各种不同视网膜疾病的广泛患者受益带来了希望。另一方面，临床研究揭示了所应用的给药策略的局限性，这可能限制其普遍使用。为此，合成基因传递平台（如基于聚合物的纳米颗粒（PNP））的玻璃体内给药已成为当前主流方法的有吸引力的替代方法。然而，为了取得成功，合成平台必须克服玻璃体内给药后在人眼中遇到的关键生物学障碍，包括玻璃体凝胶和内界膜（ILM）。在这里，我们介绍了一系列实验，从 PNP 的制备到相关实验模型的综合评估，以确定 PNP 是否克服这些障碍并有效地将治疗性基因有效载荷递送至视网膜细胞。最后，我们讨论了成功实施该策略的一些重要考虑因素。©2022Wiley Periodicals LLC. 基本方案 1：PNP 的制备和表征 基本方案 2：体外转染效率的评估 基本方案 3：PNP 在玻璃体内扩散的评估 基本方案 4：在体视培养物中外评估 PNP 穿透玻璃体视网膜外植体 基本方案 5：玻璃体内给予 PNP 介导的体内转基因表达的评估。
