Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2003) 5 OC25

BES2003 Oral Communications Brain and Behaviour (8 abstracts)

Novel methods of delivery of neurotrophic growth factor genes to CNS neurons

A Logan


Department of Medicine, University of Birmingham, Birmingham, UK.


The delivery of genes encoding neurotrophic growth factors (NFG) to injured CNS neurons promotes survival and sustains axon regeneration. The plasmid-based non-viral vectors that we have used are less toxic and safer than viruses. However, their efficiency is low, particularly in non-proliferating cells like neurons, where penetration of the nuclear membrane to access transcriptional machinery is a major factor limiting activity. We are, therefore, exploiting cytoplasmic expression systems for long-term therapeutic gene expression in CNS neurons by using mRNA therapeutically, since mRNA is expressed efficiently in the cytosol, even in quiescent cells. The system we have developed uses a plasmid encoding a NFG driven by a bateriophage T7 promoter. Polycation condensed mRNA, encoding the T7 polymerase, is co-delivered. We have shown that transfection of primary cultures of dorsal root ganglion neurons (DRGN) with lipoplex-condensed mRNA encoding a luciferase reporter produced 2 ng of enzyme per mg of cellular protein, and this will provide sufficient levels of cytoplasmic T7 RNA polymerase to drive the NFG expression in the cytoplasm. Furthermore, reporter expression was sustained for at least 5 days. The levels of T7 expressed have been further enhanced and sustained by simultaneously delivering a plasmid encoding T7, also under T7 promoter control. Thus, polymerase transcription in the cytoplasm occurs by an autoregulatory (i.e. autogene) mechanism to sustain the cytoplasmic transcription of the plasmid DNA. Using these constructs we have shown that: (1) The duration of expression of T7 polymerase protein achieved by transfection of DRGN with mRNA encoding T7 polymerase can be extended by co-transfecting with a plasmid DNA encoding T7 polymerase as an autogene. (2) Maximum expression of a T7 promoter-driven cytoplasmic reporter/therapeutic gene is achievable by co-administration of the plasmids with mRNA encoding T7, rather than by administering the mRNA first. (3) Reporter gene expression (and therapeutic gene expression) correlates with cellular levels of T7 polymerase.

Volume 5

22nd Joint Meeting of the British Endocrine Societies

British Endocrine Societies 

Browse other volumes

Article tools

My recent searches

No recent searches.