Yousefpour Parisa, Gregory Justin R, Si Kristen, Lonzaric Jan, Li Yingzhong, Wang Junmin, Qureshi Kashif, Ledbetter Amir, Lemnios Ashley A, Dye Jonathan, Remba Tanaka K, Yeung Rachel, Güereca Melissa, Rodriguez Linette, Zhang Yuebao, Wu Shengwei, Dong Yizhou, Weiss Ron, Irvine Darrell J
Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
bioRxiv. 2025 Jun 8:2025.06.08.658495. doi: 10.1101/2025.06.08.658495.
The design of gene therapies with drug-regulatable expression of therapeutic payloads is of interest for diverse applications. We hypothesized that a regulated expression system based on alphavirus-derived self-amplifying RNAs (saRNAs), which encode 4 non-structural proteins (nsPs) that copy the RNA backbone to enable sustained expression, would have advantages in safety and simplicity of delivery. Here we designed saRNAs where payload expression is regulated by the FDA-approved drug trimethoprim (TMP), by fusing TMP-responsive degradation domains (DDs) to nsPs to regulate RNA self-amplification. Screening a library of nsP-DD fusions, we identified an optimal design with DDs fused to nsP2, nsP3, and the payload, achieving a high fold-change in expression level in response to TMP and low expression in the off state. In mice, this saRNA circuit enabled diverse dynamic expression patterns in response to oral TMP. Implementing this circuit for controlled expression of an HIV antigen, an escalating TMP regimen significantly enhanced germinal center responses critical for B cell affinity maturation. This drug-regulated RNA technology holds potential for vaccines, immunotherapies, and gene therapies.
设计具有药物可调节治疗有效载荷表达的基因疗法在多种应用中具有重要意义。我们推测，基于α病毒衍生的自扩增RNA（saRNAs）的调控表达系统具有优势，该系统编码4种非结构蛋白（nsPs），可复制RNA骨架以实现持续表达，在安全性和递送简便性方面表现出色。在此，我们设计了saRNAs，通过将FDA批准的药物甲氧苄啶（TMP）响应性降解结构域（DDs）与nsPs融合以调节RNA自我扩增，从而调控有效载荷的表达。通过筛选nsP-DD融合文库，我们确定了一种最佳设计，即将DDs融合到nsP2、nsP3和有效载荷上，实现了对TMP响应的高表达水平倍数变化以及关闭状态下的低表达。在小鼠中，这种saRNA回路能够响应口服TMP实现多种动态表达模式。将该回路用于HIV抗原的可控表达，递增的TMP方案显著增强了对B细胞亲和力成熟至关重要的生发中心反应。这种药物调控的RNA技术在疫苗、免疫疗法和基因疗法方面具有潜力。
