Inflammation-Free shRNA Expression Vectors for Cystic Fibrosis Gene Therapy.

Lawton AE, Smith SE, Gill DR, Hyde SC

Human Gene Therapy, 20 4 404

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The British Society of Gene Therapy Annual Conference, London, 2009

We have recently developed CpG-free plasmid (pDNA) vectors that direct sustained, inflammation-free transgene expression when delivered to the lungs of animal models (Hyde et al, 2008 Nature Biotechnology 26:549). The delivery of such vectors expressing the CFTR epithelial chloride channel to the lungs of individuals with Cystic Fibrosis (CF) is being evaluated clinically. However CF lung pathology is also linked to excessive sodium absorption via the epithelial sodium channel ENaC. Here we describe the development of CpG-free pDNA vectors to express RNA interference (RNAi) molecules suitable for ENaC inhibition. Conventionally, such vectors utilise polIII promoters to express short hairpin RNA (shRNA) sequences. However, the high expression levels achieved by such vectors have proven toxic in some systems, limiting their duration of action (Grimm et al, 2006 Nature 441:537). To facilitate benign, long-term RNAi, we utilised the hCEFI polII enhancer/promoter element and expressed shRNA sequences embedded in a miR30 miRNA backbone. As the native miR30 backbone contains 4 CpGs, we created a CpG-free form (miR30?CpG) containing additional compensatory nucelotide changes to conserve the RNA secondary structure - essential for efficient miRNA/shRNA processing by Drosha/Dicer RNAi pathway. A version of this novel CpG-free RNAi expression vector fitted with an shRNA loop targeting luciferase, was co-transfected with a luciferase transgene expressing plasmid into HEK293T cells. Encouragingly, efficient ~80% knockdown of luciferase expression was observed (p=0.042) in these cells, along with ~60% knockdown in similar experiments using cell lines expressing luciferase constitutively (p=0.025). Subsequent vector refinements and in vivo studies will be discussed.