The DreAM-plus integrative RNA switch enhances transient AAV expression and reduces side effects of gene editing.

Uncontrolled long-term adeno-associated virus (AAV) expression prohibits therapeutic strategies that require more precise and dynamic regulation. For example, long-lasting expression of gene editors by AAV could augment off-target effects and immunogenicity. Drug-inducible RNA switches are desirable tools to achieve transient AAV expression. However, current RNA switches only target a single mechanism such as transcription or RNA splicing, exhibiting limited capacity in transgene regulation. Here, we report DreAM-plus, a multilayer RNA switch that integrates an aptamer-based poly(A) regulator (pA), a drug-elicitable alternative splicing module (DreAM), and an engineered P2A element with conditional upstream open reading frames (uORFs). The pA-DreAM concatenation enhanced gene inducibility by up to 5-fold more than pA or DreAM alone, with 1.4- to 6.3-fold further improvement by uORFs. DreAM-plus achieved transient expression of an array of gene editors (SpCas9, SaCas9, Un1Cas12f1, OsCas12f1, AcCas12n, and IsDra2 TnpB) with a temporal resolution of less than 24 h, which significantly mitigated off-target effects by 1.4- to 2.8-fold. With lipid-nanoparticle-delivered pre-existing immunity in mice, DreAM-plus attenuated AAV-delivered Cas-specific CD8+ T cell immune toxicity in the liver and heart. Therefore, the inducible RNA switches could be synergistically integrated to build sophisticated genetic cassettes for enhanced safety of AAV-mediated gene editing.