Embedding siRNA sequences targeting Apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy

• Published in: Molecular therapy Vol. 21; no. 1; pp. 217 - 227
• Main Authors: Maczuga, Piotr, Lubelski, Jacek, van Logtenstein, Richard, Borel, Florie, Blits, Bas, Fakkert, Erwin, Costessi, Adalberto, Butler, Derek, van Deventer, Sander, Petry, Harald, Koornneef, Annemart, Konstantinova, Pavlina
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2013; Elsevier Limited; Nature Publishing Group
• Subjects: Adeno-associated virus; Animals; Apolipoprotein B-100 - genetics; Apolipoproteins; Base Sequence; Blotting, Western; Cardiovascular disease; Cholesterol; Cholesterol - blood; Gastroenterology; Gene expression; Gene Knockdown Techniques; Genetic Vectors; Genomes; HEK293 Cells; Hepatology; Humans; Liver; Liver - metabolism; Male; Mice; Mice, Inbred C57BL; MicroRNAs; MicroRNAs - chemistry; MicroRNAs - genetics; Original; Polymerase Chain Reaction; R&D; Research & development; RNA Processing, Post-Transcriptional; RNA, Small Interfering - chemistry; RNA, Small Interfering - genetics; Toxicity; Netherlands
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1038/mt.2012.160

Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5′ and 3′ cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 1011 genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics.