Transduction of COS-7 and K562 cell lines by recombinant lentiviral particles carrying miniLCR and B-globin gene: Introduction to gene therapy of major B-thalassemia

ABSTRACT

Beta-thalassemia is caused by absence of reduction of beta-globin chain synthesis. One of the effective therapeutic methods for this disease can be gene therapy by viral vectors. The capacity of lentiviral vectors is approximately 8 kb, we designed a 6 kb construct containing mini LCR and beta-globin gene instead of LCR region. The aim of this study is to make a recombinant lentiviruses containing miniLCR and beta-globin gene for transfer to the target cells for gene therapy of beta-thalassemia. HS2, HS3, HS4 segments [miniLCR] and beta-globin gene with 5' and 3' UTR were amplified from the genomic DNA of a normal individual by PCR. Each segment was cloned in pTZ57R/T vector and then sub cloned first into the pBGGT vector and finally into the pLenti-Dest vector. Final transfer vector and the three helper packaging plasmids [Plp1, Plp2 Plp/VSVG] were contransfected 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 determined in a K562 cell line and compared with COS-7 cell line. The titer in both cell lines was the same. Optimum MOI for COS-7 cell line was 5 and when polybrene was used transduction increased by 2 fold. The remaining transduced COS-7 colonies were expanded and DNA was extracted. By PCR, random intergration construct into the genome was evaluated. The produced lentiviruses can be an appropriate means for effective transfer of the designed construct into dividing and non-dividing cells such as hematopoetic stem cells for transplantation of beta thalassemia patients. Efficiency of transduction by leniviruses is more than the gene targeting technique. Also units of HS2, HS3 and HS4 regions in mini LCR and selection of larger HS3 unit may increase the expression of beta globin gene