RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes

Adeno-associated virus (AAV)-based gene therapy could be facilitated by the development of molecular switches to control the magnitude and timing of expression of therapeutic transgenes. RNA interference (RNAi)-based approaches hold unique potential as a clinically proven modality to pharmacologically regulate AAV gene dosage in a sequence-specific manner. We present a generalizable RNAi-based rheostat wherein AAV transgene expression is silenced using the clinically validated modality of chemically modified short interfering RNA (siRNA) conjugates or vectorized co-expression of short hairpin RNA (shRNA). For transgene induction, we employ REVERSIR technology, a synthetic high-affinity oligonucleotide complementary to the siRNA or shRNA guide strand to reverse RNAi activity and rapidly recover transgene expression. For potential clinical development, we report potent and specific siRNA sequences that may allow selective regulation of transgenes while minimizing unintended off-target effects. Our results establish a conceptual framework for RNAi-based regulatory switches with potential for infrequent dosing in clinical settings to dynamically modulate expression of virally-delivered gene therapies.基于腺相关病毒（Adeno-associated virus, AAV）的基因治疗，可通过开发分子开关来精准调控治疗性转基因表达的强度与时序。基于RNA干扰（RNA interference, RNAi）的方法作为经临床验证的技术手段，在以序列特异性方式药理学调控AAV基因剂量方面具备独特潜力。我们报道了一种可通用的基于RNA干扰的表达调控变阻器：可通过经临床验证的化学修饰短干扰RNA（short interfering RNA, siRNA）偶联物，或是载体介导的短发夹RNA（short hairpin RNA, shRNA）共表达的方式，沉默AAV的转基因表达。在诱导转基因表达时，我们采用了REVERSIR技术——一种可与siRNA或shRNA向导链互补的合成高亲和性寡核苷酸，能够逆转RNA干扰活性并快速恢复转基因表达。针对潜在的临床转化应用，我们报道了一系列强效且特异性的siRNA序列，可实现转基因的选择性调控，同时最大限度减少非预期的脱靶效应。本研究结果为基于RNA干扰的调控开关构建了概念框架，该框架有望在临床场景中通过低频给药，动态调控病毒递送的基因治疗药物的表达。