RESUMEN
Genetic modification of hematopoietic stem cells holds great promise in the treatment of hematopoietic disorders. However, clinical application of gene delivery has been limited, in part, by low gene transfer efficiency. To overcome this problem, we investigated the effect of retronectin (RN) on lentiviral-mediated gene delivery into hematopoietic progenitor cells (HPCs) derived from bone marrow both in vitro and in vivo. RN has been shown to enhance transduction by promoting colocalization of lentivirus and target cells. We found that RN enhanced lentiviral transfer of the VENUS transgene into cultured c-Kit(+) Lin(-) HPCs. As a complementary approach, in vivo gene delivery was performed by subjecting mice to intra-bone marrow injection of lentivirus or a mixture of RN and lentivirus. We found that co-injection with RN increased the number of VENUS-expressing c-Kit(+) Lin(-) HPCs in bone marrow by 2-fold. Further analysis of VENUS expression in colony-forming cells from the bone marrow of these animals revealed that RN increased gene delivery among these cells by 4-fold. In conclusion, RN is effective in enhancing lentivirus-mediated gene delivery into HPCs.
Asunto(s)
Fibronectinas/farmacología , Técnicas de Transferencia de Gen , Células Madre Hematopoyéticas/efectos de los fármacos , Lentivirus/genética , Proteínas Recombinantes/farmacología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células Cultivadas , Evaluación Preclínica de Medicamentos , Fibronectinas/química , Células Madre Hematopoyéticas/metabolismo , Humanos , Lentivirus/fisiología , Ratones , Ratones Endogámicos C57BL , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/metabolismo , Fragmentos de Péptidos/farmacología , Estructura Terciaria de Proteína , Regulación hacia ArribaRESUMEN
A 25-kDa linear polyethylenimine (25 kDa L-PEI) has proven to be efficient and versatile agent for gene delivery. Therefore, we determined the optimal transfection conditions of 25 kDa L-PEI and examined whether it has comparable transfection efficiency with other commercially available reagents, ExGen 500, LipofectAMINE 2000, and Effectene by using EGFP expression vector in different cell lines. Transfection efficiency and cytotoxicity were measured by flow cytometry. First of all, we determined the optimal ratio of nitrogen to phosphorous (N/P) and DNA concentration. With the increase of N/P ratio and DNA amounts, transfection efficiency increased with a slight variation in cell types. The optimal amounts of 25 kDa L-PEI were determined at N/P ratio 40 and DNA concentration varied among the cell types. In addition, 25 kDa L-PEI worked efficiently and was less toxic than other reagents. However, the efficiency and toxicity of all these reagents varied according to cell types as well as the ratio of DNA to reagents and the amounts of DNA. Our finding illustrates the importance of optimal transfection conditions of 25 kDa L-PEI to obtain maximal transgene expression with less cytotoxicity. Importantly, the optimization of those conditions may make possible to perform transfection cost-effectively and efficiently.