RNAi and CRISPR/Cas9 Based In Vivo Models for Drug Discovery

With the advent of RNAi and more recently CRISPR/Cas9 technologies, we now have an advanced platform to rapidly generate disease and perform target validation and toxicity assessment of novel candidate targets all in the same animal. Having previously engineered a miRE scaffold for enhanced shRNA processing and now with the introduction of a powerful new SplashRNA algorithm for accurate shRNA prediction, we have brought RNAi technologies to its peak, by increasing potency and reducing off-target effects, such that it can be effectively exploited to preclinically mimic drug therapy and even combination therapy not only in vitro but also in live mice. Here, we take advantage of doxycycline-inducible RNAi mice to identify on-target toxicities associated inhibition of Rpl15 ,a novel gene target previously identified through screening as a potent therapeutic target for the treatment of HCC. We demonstrate that sustained Rpl15 suppression results in an impaired intestinal barrier with subsequent weight loss. Heterozygous mutation or deletion of Rpl15 has been associated with Diamond Blackfan Anemia in humans and we therefore analyzed our mice for hematological phenotypes. Peripheral blood counts revealed only a non-significant decrease in erythrocytes and hemoglobin in Rpl15 knockdown mice compared to mice expressing a control shRNA. However, we found that Rpl15 suppression resulted in a strong decrease in spleen size and histopathological analyses revealed signs of increased siderosis. Importantly, phenotypic toxicities are not observed following doxycycline removal and reexpression of Rpl15, thus suggesting that a therapeutic window of treatment exists for inhibition of this essential gene. By combining this approach with in situ delivery of Cas9/gRNAs, we proceed one step further to rapidly develop HCC and validate Rpl15 as potent target of HCC in vivo . These data showcase how synergizing our RNAi and CRISPR/Cas9 genetic toolbox will help facilitate drug discovery research and increase our confidence in predicting drug responses in humans into a new era.