Antisense gene delivered by an adenoassociated viral vector inhibits iron uptake in human intestinal cells: potential application in hemochromatosis.
Hereditary hemochromatosis (HH) is a condition in which intestinal iron absorption is greatly elevated. Present treatment is weekly phlebotomy, affecting quality of life and leading to recurrent infections. The iron transporter divalent metal transporter-1 (DMT-1) of enterocytes is responsible for iron uptake from the intestinal lumen; iron is further extruded into the blood by the basolateral transporter ferroportin-1. A therapeutic approach for HH could start with a long-term reduction of iron transport by reduction of DMT-1 levels. We designed an AAV vector coding for a short antisense RNA (AAV-DMT-1-AS) against DMT-1, which reduced iron uptake by 50-60% in human intestinal cells (Caco-2). At low infection levels, DMT-1 mRNA virtually disappeared, suggesting RNAi-like and/or RNase H antisense effects. DMT-1 mRNA levels returned to normal at higher infection levels, indicating that an additional mechanism of mRNA occupation, able to block DMT-1 translation and to avoid feedback regulation by iron responsive elements (IRE), also exists. Cell morphology was normal in all cases and no increases in the interferon-related responses, measured by (a) 2'-5' A oligo synthetase (b) IFITM1 and (c) ISGF3gamma mRNA levels, were observed. Studies presented herein indicate that enterocyte targeting with a gene coding for a short antisense against iron transport blocks enterocyte iron uptake, which may have therapeutic value.
Ezquer
Fernando
F
Millennium Institute for Advanced Studies in Cell Biology and Biotechnology, Chile.
Núñez
Marco Tulio
MT
Israel
Yedy
Y
D018938:Caco-2 Cells; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D006432:Hemochromatosis / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000187:drug effects; D007501:Iron / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016372:RNA, Antisense / Q000008:administration & dosage* / Q000235:genetics
D016372:RNA, Antisense; D007501:Iron