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

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

Insulin receptor gene mutations in Iranian patients with type II diabetes mellitus

Patients with diabetes mellitus type II suffer from hyperglycemia because they are not able to use the insulin that they produce, often due to inadequate function of insulin receptors. There are some evidences that this deficiency is inherited in a dominant autosomal manner and leads to the malfunction of the pancreatic beta cells resulting in insulin excretion disorders. In this study, we sought to identify mutations in the insulin receptor [INSR] gene, which can cause insulin resistance in type II diabetic patients. DNA was extracted from peripheral blood cells of the patients [n = 128] diagnosed with type II diabetes. All 22 exons of the INSR gene of the patients were analyzed for mutations running PCR, conformation-sensitive gel electrophoresis and DNA sequencing, consecutively. Approximately 26% of the patients had genetic mutations; however, most of them were not reported. These mutations include exon 2 [His171Asn, Ile172Ser, Cys196Ser and Ser210Arg], exon 3 [Gly227Asp and Gly232Ser], exon 8 [Thr543Ser], exon 9 [a heterozygote was observed with no change in phenylalanine at position 669], exon 13 [two heterozygotes: Arg890Pro with Asn865 remaining unchanged], exon 14 [Ala906Gly and Pro918Trp with Arg902 unchanged], exon 17 [Val1086Glu] and exon 19 [His1157Gln with Thr1172 unchanged]. The lack of similar mutation records in literature and genetic data banks may suggest a geographic pattern for these INSR gene variants in our population

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

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