Circular RNAs (circRNAs) are long-lived, covalently closed RNAs that are abundantly expressed and evolutionarily conserved across eukaryotes. Possible functions ranging from microRNA (miRNA) and RNA binding protein sponges to regulators of transcription and translation have been proposed. Here we describe the design and characterization of recombinant adeno-associated viral (AAV) vectors packaging transgene cassettes containing intronic sequences that promote backsplicing to generate circularized RNA transcripts. Using a split GFP transgene, we demonstrate the capacity of vectors containing different flanking intronic sequences to efficiently drive persistent circRNA formation in vitro. Further, translation from circRNA is efficiently driven by an internal ribosomal entry site (IRES). Upon injecting AAV vectors encoding circRNA in mice, we observed robust transgene expression in the heart, but low transduction in the liver for the intronic elements tested. Expression in the murine brain was restricted to astrocytes following systemic or intracranial administration, while intravitreal injection in the eye yielded robust transgene expression across multiple retinal cell layers. These results highlight the potential for exploiting AAV-based circRNA expression to study circRNA function and tissue-specific regulation in animal models, as well as development of therapeutic platforms using this approach.

环状 RNA (circRNA) 是长寿命、共价闭合的 RNA，在真核生物中大量表达且进化保守。已经提出了从 microRNA (miRNA) 和 RNA 结合蛋白海绵到转录和翻译调节因子的可能功能。在这里，我们描述了重组腺相关病毒 (AAV) 载体的设计和表征，该载体包装了含有内含子序列的转基因盒，这些内含子序列可促进反向剪接以生成环化 RNA 转录本。使用分裂的 GFP 转基因，我们证明了包含不同侧翼内含子序列的载体在体外有效驱动持久 circRNA 形成的能力。此外，circRNA 的翻译是由内部核糖体进入位点 (IRES) 有效驱动的。在向小鼠注射编码 circRNA 的 AAV 载体后，我们观察到心脏中的转基因表达强劲，但测试的内含子元件在肝脏中的转导较低。全身或颅内给药后，小鼠大脑中的表达仅限于星形胶质细胞，而眼部玻璃体内注射则在多个视网膜细胞层中产生了强劲的转基因表达。这些结果凸显了利用基于 AAV 的 circRNA 表达来研究动物模型中的 circRNA 功能和组织特异性调控的潜力，以及使用这种方法开发治疗平台的潜力。