Reversal of Mdr1b-dependent Multidrug Resistance in a Rat Astrocyte Model by Adenoviral-delivered Short Hairpin RNA

Abstract

Over-expression of P-glycoprotein (Pgp), a protein responsible for multidrug resistance (MDR), is responsible for general resistance to anti-epileptic drugs (AEDs). We explored the potential use of gene therapy with adenoviral-delivered RNA interference against mdr1b as a method to sensitize refractory epilepsy to AEDs. We constructed replication-deficient recombinant adenovirus Adeno-mdr1b1 carrying short hairpin RNA (shRNA) targeting against mdr1b, and successfully infected the established Sprague-Dawley rat astrocyte model of Coriaria Lactone-induced Pgp over-expression. The expression levels of mdr1b and Pgp and the Rhodamine123 efflux ratio in trial groups were significantly lower than that of blank control (P \ 0.05) during the first 7 days post-infection, with the most inhibition at 48 h. The results suggest that knockdown of MDR using adenovirus not only avoided the toxicity and low rate of plasmid nucleofection, but also overcame its poor efficiency of mdr1b silencing.More importantly, this study may pave the way for a promising approach to remedy refractory epilepsy.

Applications of iRNA Technologies in Drug Transporters and Drug Metabolizing Enzymes

“…Imaging Pgp transport activity and inhibition in a variety of tissues within the intact animal and direct documentation of shRNAi -targeted effects in vivo using bioluminescence provide further evidence to establish the potential of knockdown strategies for circumventing MDR. Chen et al ( 2008Chen et al ( , 2009 ) explored the potential use of gene therapy with adenoviral -delivered RNAi against mdr1b as a method to sensitize refractory epilepsy to antiepileptic drug s ( AED s). They constructed replication -defi cient recombinant adenovirus Adenomdr1b1 carrying shRNA targeting against mdr1b, and successfully infected the established Sprague Dawley rat astrocyte model of coriaria lactone -induced Pgp over -expression.…”

RNAi Inhibits Coriaria Lactone-Induced MDR1b Overexpression in Rat Brain Microvascular Endothelial Cells

“…For exact construction, amplification, and purification, please refer to the study of Chen et al (2008). The final virus titers were: Ad5-enhanced green fluorescence protein (EGFP): 8.5× 10 11 v.p.…”

Astrocytic Responses to DNA Delivery Using Nucleofection

“…Viral transduction methods have generally provided higher efficiencies and lower cytotoxicity. For example, retroviral transduction of astrocytes in primary culture typically results in more than 80% transgene expression [1,2], while adenoviruses generally produce a lower expression efficiency of 30-50% [3,4]. Later generation adeno-associated viruses have given expression rates up to 90% in mixed glialneuronal primary cultures accompanied by low toxicity [5].…”

“…This technology has been applied successfully to transfect several cell types such as primary neurons that are hard to transfect using other non-viral methods [17,18]. Although procedures have been developed for nucleofection of astrocytes grown in culture by the manufacturer, so far only a few studies have used this approach to transfect these cells [3,19].…”