Inflammation-Free shRNA Expression Vectors for Cystic Fibrosis Gene Therapy. (2009)

Lawton, A. E., Smith, S. E., Gill, D. R. & Hyde, S. C.

Human Gene Therapy, 20, 404

Download   Back

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.

Home
News
Introductory Videos
Medical Futures Innovation Award 2011
Twitter Feed
About Us
Contact Us
Careers
Resources
Lab Events
Environemental Policy
About this Site

Google Site Search

Site Feedback Form

All Site Images

 

UK CFGTC

How the Consortium works/FAQs

Consortium Website

Centre for Molecular Medicine, Edinburgh
The Roslin Institute
Dep of Gene Therapy, Imperial

Milestones

GL67A/pGM169

The Run-in Study

Single Dose Clinical Trial

Multi Dose Clinical Trial

 

Our Research

Non-viral Vector Development

Aerosol Mediated Gene Delivery

Viral Vector Development

Taqman Core Facility

Cystic Fibrosis

History of CF

Discovery of the CFTR Gene

CFTR Protein Structure

CFTR Function

CF Links

 

Gene Therapy

Introduction to Gene Therapy

Other CF Gene Therapy Groups

Why use Gene Therapy for CF

Target Cells for CF Gene Therapy

Barriers for CF Gene Therapy

Clinical Trials

Gene Therapy Successes

Gene Therapy Links

 

 

Publications

Papers in Journals

Conference Posters & Presentations

Book Chapters

D.Phil Theses

Lectures

 

Gene Therapy Seminars

Schedule

Directions & Venue