Drug Discovery
more natural process where the passenger strand is degraded almost 100% of the time, helping to increase the potency of the shRNAs and avoid off- targets caused by the passenger strand19. Now we have even progressed beyond our third- generation miR30 structures and developed miR- E, the most effective scaffold for RNAi-mediated gene silencing19,20. With this miRNA backbone, we see effective gene silencing even with only a sin- gle copy genomic integration, which also dramati- cally decreases your chances for off-target effects. With this new structure, we can also express shRNAs in tandem, enabling potent inhibition of multiple gene targets simultaneously. By using this approach we can mirror drugs that inhibit protein families rather than single enzymes, providing an avenue for critical preclinical evaluation of multi- target inhibition or combination therapies. Our group also spent about a decade building data sets (more than 500,000 shRNAs) and com- piled them, in collaboration with Christina Leslie’s group at Memorial Sloan Kettering Cancer Center, into a sequential learning algorithm called SplashRNA that allows researchers to predict the best shRNAs for a given gene of interest with a high degree of certainty18,21. This dramatically reduces the amount of screening needed to predict microRNA-based shRNAs for many genes. Once we identify a good shRNA sequence we can use them to create mice in as little as three months. When combined with the tetracycline inducible system22, expression of shRNAs can be controlled
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by treating mice with doxycycline (a tetracycline analog) in their food or drinking water, which will induce gene silencing. Removal of doxycycline will reverse the system and cause gene re-expression to endogenous levels in four to seven days23. A study of acute myeloid leukaemia nicely illus- trates the utility of RNAi in mimicking therapy. The researchers conducting the study wanted to determine whether epigenetic regulators of an AML cell line they had created might be potential targets for drugs24. Armed with 300 genes and a library of about 1,000 shRNAs targeting those genes, they added viral particles encoding pooled shRNA DNA to the AML cells, cultured them and then evaluated which shRNAs were lost after cul- turing – potential evidence that specific gene inhi- bition had killed the AML cell lines. From this experiment, the group identified the protein bro- modomain-containing 4 (Brd4) as being critically required for disease maintenance and its inhibition as a vulnerability in tumour growth. The researchers found that both suppression of Brd4 using shRNAs or using JQ1, a potent inhibitor of the BET bromodomain family (BRD2, BRD3, BRD4 and BRDT)25 had a robust effect on disease progression both in vitro and in vivo. These results are one of dozens illustrating the ability of RNAi to mimic drug therapy.
Following these results, using RNAi mice con- taining a tet-inducible Brd4 shRNA, Scott Lowe’s group at MSKCC was able to show that suppres- sion of Brd4 alters normal hematopoiesis, causes
Drug Discovery World Fall 2017
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