基因治疗是治疗遗传疾病的一种突破性方法。人们希望通过病毒或非病毒载体将外源基因导入活细胞来实现基因修复、基因替代或基因表达调控，达到治疗遗传疾病的目的。

为了能够尽量减少病毒中和抗体的影响，为重复给药提供可能。近期，Ring Therapeutics公司开发出一种新型病毒载体——指环病毒（Anellovirus）。

指环病毒是一类无囊膜、负链、环状单链DNA（ssDNA）病毒，能够感染脊椎动物，广泛纯在于人的血液及多数组织和器官中⁵。重要的是指环病毒能够逃避诱导体液免疫反应，且似乎不具致病性⁶。这些特性，结合其庞大的基因组多样性，使指环病毒成为极具吸引力的潜在基因药物递送载体。

作者通过开发的SATURN系统能够实现高效的病毒生产，并在多种组织类型中成功实现基因递送，且具有与AAV类似的递送效果。这一成果不仅验证了指环病毒作为一种新型基因递送平台的潜力，也为未来基因治疗领域的研究和应用提供了新的方向。

Viral harvest

培养细胞裂解：MOLT-4 cells were pelleted by centrifugation and then resuspended in lysis buffer containing 50 mM Tris pH 8.0, 0.5% Triton-X100, 100 mM NaCl, 1 × Halt protease inhibitor cocktail, 50 mM Tris pH 8.0, and 2 mM MgCl2 and 200 U of mSAN nuclease. Cell lysates were incubated at 37°C for 1 hour. Cell lysates were then clarified at 10,000 × g for 20 minutes at 4°C to pellet cellular debris.

密度梯度离心：Iodixanol linear gradients

To generate iodixanol linear gradients, 13 mL of 60% OptiPrep was layered over with 13 mL of 20% OptiPrep in 26.3-mL polycarbonate tubes. These tubes were subsequently spun at a 46-degree angle at a speed of 20 rpm for 16 minutes using a Gradient Master (BioComp). The sample tubes were then centrifuged at 347,000 × g and 20°C for 3 hours using a Type 70 Ti rotor (Beckman Coulter). 1-mL fractions were collected from the top of the tube. Each fraction underwent a DNase protected qPCR assay, following the procedure outlined below.