Jantje Gerdes, Evotec International, Göttingen

Compared to small molecule modalities, testing RNA-interference based therapeutics in vivo faces unique challenges. For efficient knockdown, the siRNA must be a complete match to the targeting sequence. However, often the sequence homology between rodent species such as rat or mouse is not high enough to use the same sequence in preclinical animal models and clinical studies leading to the need for additional testing strategies.

Here, we present an approach to identify the most effective GalNAc-modified siRNAs for knockdown of human targets in the mouse liver.

Briefly, we overexpressed fragments of the human target gene in murine liver using a viral delivery vector AAV DJ/8. After two weeks, we applied different GalNAC-modified siRNAs and checked for target gene expression four weeks after that. We found that the coding sequence of two different genes was generally more effectively suppressed than fragments of the 3’ untranslated region of the same genes. In addition, we observed hot spots within the mRNA sequence that were more efficiently targeted than other regions of the nucleotide sequence. We identified several siRNAs that achieved more than 80% knockdown of the human target gene sequence after four weeks. This approach demonstrates how transient overexpression of human target structures leads to the identification of potent siRNA target sequences for treatment of human disease.