Roychoudhury Pavitra, De Silva Feelixge Harshana S, Pietz Harlan L, Stone Daniel, Jerome Keith R, Schiffer Joshua T
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
J Antimicrob Chemother. 2016 Aug;71(8):2089-99. doi: 10.1093/jac/dkw104. Epub 2016 Apr 18.
A promising curative approach for HIV is to use designer endonucleases that bind and cleave specific target sequences within latent genomes, resulting in mutations that render the virus replication incompetent. We developed a mathematical model to describe the expression and activity of endonucleases delivered to HIV-infected cells using engineered viral vectors in order to guide dose selection and predict therapeutic outcomes.
We developed a mechanistic model that predicts the number of transgene copies expressed at a given dose in individual target cells from fluorescence of a reporter gene. We fitted the model to flow cytometry datasets to determine the optimal vector serotype, promoter and dose required to achieve maximum expression.
We showed that our model provides a more accurate measure of transduction efficiency compared with gating-based methods, which underestimate the percentage of cells expressing reporter genes. We identified that gene expression follows a sigmoid dose-response relationship and that the level of gene expression saturation depends on vector serotype and promoter. We also demonstrated that significant bottlenecks exist at the level of viral uptake and gene expression: only ∼1 in 220 added vectors enter a cell and, of these, depending on the dose and promoter used, between 1 in 15 and 1 in 1500 express transgene.
Our model provides a quantitative method of dose selection and optimization that can be readily applied to a wide range of other gene therapy applications. Reducing bottlenecks in delivery will be key to reducing the number of doses required for a functional cure.
一种有前景的治疗艾滋病病毒（HIV）的方法是使用定制的核酸内切酶，该酶可结合并切割潜伏基因组内的特定靶序列，从而产生使病毒无法复制的突变。我们开发了一个数学模型，用于描述使用工程化病毒载体递送至HIV感染细胞的核酸内切酶的表达和活性，以指导剂量选择并预测治疗效果。
我们开发了一个机制模型，该模型可根据报告基因的荧光预测在给定剂量下单个靶细胞中表达的转基因拷贝数。我们将该模型与流式细胞术数据集进行拟合，以确定实现最大表达所需的最佳载体血清型、启动子和剂量。
我们表明，与基于门控的方法相比，我们的模型能更准确地衡量转导效率，基于门控的方法会低估表达报告基因的细胞百分比。我们确定基因表达遵循S形剂量反应关系，并且基因表达饱和水平取决于载体血清型和启动子。我们还证明在病毒摄取和基因表达水平存在显著瓶颈：添加的载体中只有约1/220进入细胞，在这些细胞中，根据所用剂量和启动子的不同，每15至1500个细胞中有1个表达转基因。
我们的模型提供了一种剂量选择和优化的定量方法，可轻松应用于广泛的其他基因治疗应用。减少递送过程中的瓶颈将是减少功能性治愈所需剂量数量的关键。
