Chinnasamy Dhanalakshmi, Fairbairn Leslie J, Neuenfeldt James, Treisman Jonathan S, Hanson John P, Margison Geoffrey P, Chinnasamy Nachimuthu
Vince Lombardi Gene Therapy Laboratory, Immunotherapy Program, St. Luke's Medical Center, Milwaukee, WI 53151, USA.
Hum Gene Ther. 2004 Aug;15(8):758-69. doi: 10.1089/1043034041648417.
Lentiviral vectors are capable of efficiently transducing nondividing and slowly dividing cells, including hematopoietic stem cells, resulting in stable integration and sustained transgene expression. We constructed human immunodeficiency virus type 1-based self-inactivating lentiviral vectors to express either wild-type or an O6-benzylguanine (O6-beG)-resistant mutant form of the human O6-alkylguanine-DNA methyltransferase (MGMT; DNA-O6-methylguanine:[protein]-L-cysteine S-methyltransferase, EC 2.1.1.63) and transduced K562 and granulocyte colony-stimulating factor-mobilized human peripheral blood CD34+ cells. After transduction, K562 cells expressed high levels of MGMT as determined by Western blot, immunocytochemistry, and biochemical assay. A colony-forming survival assay showed significant protection against O6-beG plus 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) or temozolomide (TMZ) toxicity. Similarly, a single transduction of CD34+ cells resulted in a 13- to 14-fold increase in the level of MGMT expression. In comparison with non-transduced cells, mutant MGMTP140K-transduced CD34+ cells showed significant resistance against the combined toxicity of O6-beG with either TMZ or BCNU: there was an approximately 9-fold increase in the survival of colony-forming cells as indicated by the IC50 values after O6-beG plus TMZ treatment and an approximately 5-fold increase in the case of O6-beG plus BCNU treatment. These results show that lentivirus-mediated expression of MGMTP140K can efficiently protect the hematopoietic compartment against the combined toxicity of O6-beG plus TMZ or BCNU.
慢病毒载体能够有效地转导非分裂和缓慢分裂的细胞，包括造血干细胞，从而实现稳定整合和持续的转基因表达。我们构建了基于1型人类免疫缺陷病毒的自失活慢病毒载体，以表达野生型或O6-苄基鸟嘌呤（O6-beG）抗性突变形式的人类O6-烷基鸟嘌呤-DNA甲基转移酶（MGMT；DNA-O6-甲基鸟嘌呤：[蛋白质]-L-半胱氨酸S-甲基转移酶，EC 2.1.1.63），并转导K562细胞和粒细胞集落刺激因子动员的人类外周血CD34+细胞。转导后，通过蛋白质印迹、免疫细胞化学和生化分析确定，K562细胞表达高水平的MGMT。集落形成存活试验显示，对O6-beG加1,3-双（2-氯乙基）-亚硝基脲（BCNU）或替莫唑胺（TMZ）毒性有显著保护作用。同样，单次转导CD34+细胞导致MGMT表达水平提高13至14倍。与未转导的细胞相比，突变型MGMTP140K转导的CD34+细胞对O6-beG与TMZ或BCNU联合毒性表现出显著抗性：O6-beG加TMZ处理后，集落形成细胞的存活率根据IC5o值增加约9倍；O6-beG加BCNU处理时，存活率增加约5倍。这些结果表明，慢病毒介导的MGMTP140K表达可有效保护造血系统免受O6-beG加TMZ或BCNU联合毒性的影响。
