[HIV type 1-based particles effect in transduction of HT1080 cell line: a technique in beta-thalassemia gene therapy]

ABSTRACT

One of the most effective methods in the treatment of beta-thalassemia is gene therapy by viral vectors. The aim of this study was to design a recombinant lentivirus containing mini LCR and beta-globin gene for transferring normal beta-globin gene into hematopoetic stem cells. In this basic-applied study, each segment was cloned into a lenti transfer vector and confirmed by restriction digestion and sequencing. Transfer vector and three packaging plasmids were cotransfected into 293T packaging cells using lipofectamine 2000. Harvested viruses were confirmed by RT PCR on extracted RNA of these recombinant lentiviruses. The titer of lentiviral stock was determined in a HT1080 cell line. Transduction of target cells was increased by polybrene until 2 fold. Transduced HT1080 colonies remained after 2-week antibiotic selection. The remained transduced HT1080 colonies were expanded and DNA was extracted. PRC evaluated random integration of construct into the genome in this gene transfer technique. PCR evaluated random integration of construct into the genome in this gene transfer technique. Optimum MOI for HT1080 cell line was determined. Lenti viruses can be used for effective and permanent gene transferring in mammalian cells such as hematopoetic stem cells in order to accomplish gene therapy of genetic diseases like beta thalassemia and cancers