Ram Z, Culver K W, Walbridge S, Blaese R M, Oldfield E H
Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke Clinical Center, NIH, Bethesda, Maryland 20892.
Cancer Res. 1993 Jan 1;53(1):83-8.
Gene transfer with vectors derived from murine retroviruses is restricted to cells which are proliferating and synthesizing DNA at the time of infection. This suggests that retroviral-mediated gene transfer might permit targeting of gene integration into malignant cells in organs composed mainly of quiescent nonproliferating cells, such as in the brain. Accordingly, selective introduction of genes encoding for susceptibility to otherwise nontoxic drugs ("suicide" genes) into proliferating brain tumors may be used to treat this cancer. We investigated the efficacy and dynamics of in vivo transduction of growing brain tumors with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir. Ganciclovir is phosphorylated by thymidine kinase to toxic triphosphates that interfere with DNA synthesis, resulting in the preferential death of the transduced tumor cells. Rats inoculated with 4 x 10(4) 9L gliosarcoma cells into the frontal lobe were treated 7 days later with an intratumoral stereotaxic injection of murine fibroblasts (NIH 3T3 cells) that were producing a retroviral vector containing the herpes simplex-thymidine kinase gene. Controls received vector producer and nonproducer NIH 3T3 cell lines containing the Escherichia coli lacZ (beta-galactosidase) gene as well as nonproducer NIH 3T3 cells containing the thymidine kinase gene. The animals were rested for 7 days to allow time for in situ transduction of the proliferating tumor cells with the herpes-thymidine kinase retroviral vector. The animals were then treated with ganciclovir, 15 mg/kg i.p. twice a day for 14 days. Gliomas receiving an injection of 3-5 x 10(6) thymidine kinase producer cells regressed completely in 23 of 30 rats given ganciclovir therapy, while 25 of 26 control rats developed large tumors. Intratumoral injection of a lower concentration of thymidine kinase vector producer cells (1.8 x 10(6)) resulted in a lower frequency of tumor regression (5 of 13 rats). To estimate the efficiency of in vivo gene transfer, 9L brain tumors were given injections of 5 x 10(6) beta-galactosidase vector producer cells. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranaside staining revealed maximal staining of beta-galactosidase within the tumor 7-14 days after injection of the vector producer cells. In vivo transduction rates in harvested tumors ranged from 10 to 70%. There was no evidence of transduction of the surrounding normal neural tissue. Occasional blood vessel endothelial cells within or adjacent to the tumor were observed to be 5-bromo-4- chloro-3-indolyl-beta-D-galactopyranaside positive.(ABSTRACT TRUNCATED AT 400 WORDS)
利用源自鼠逆转录病毒的载体进行基因转移，仅限于在感染时正在增殖并合成DNA的细胞。这表明逆转录病毒介导的基因转移可能允许将基因整合靶向到主要由静止的非增殖细胞组成的器官中的恶性细胞，比如大脑。因此，将编码对原本无毒药物敏感的基因（“自杀”基因）选择性导入增殖性脑肿瘤中，可用于治疗这种癌症。我们研究了用单纯疱疹病毒胸苷激酶基因对生长中的脑肿瘤进行体内转导的效果和动态变化，随后给予抗病毒药物更昔洛韦。更昔洛韦被胸苷激酶磷酸化为有毒的三磷酸盐，干扰DNA合成，导致转导的肿瘤细胞优先死亡。将4×10⁴个9L胶质肉瘤细胞接种到额叶的大鼠，7天后接受瘤内立体定向注射产生含有单纯疱疹病毒胸苷激酶基因的逆转录病毒载体的鼠成纤维细胞（NIH 3T3细胞）。对照组接受含有大肠杆菌lacZ（β-半乳糖苷酶）基因的载体产生细胞系和非载体产生细胞系，以及含有胸苷激酶基因的非载体产生NIH 3T3细胞。让动物休息7天，以便有时间用疱疹胸苷激酶逆转录病毒载体对增殖的肿瘤细胞进行原位转导。然后给动物腹腔注射更昔洛韦，剂量为15mg/kg，每天两次，共14天。接受注射3 - 5×10⁶个胸苷激酶产生细胞的胶质瘤，在接受更昔洛韦治疗的30只大鼠中有23只完全消退，而26只对照大鼠中有25只长出大肿瘤。瘤内注射较低浓度的胸苷激酶载体产生细胞（1.8×10⁶个）导致肿瘤消退频率较低（13只大鼠中有5只）。为了评估体内基因转移的效率，给9L脑肿瘤注射5×10⁶个β-半乳糖苷酶载体产生细胞。5-溴-4-氯-3-吲哚-β-D-吡喃半乳糖苷染色显示，在注射载体产生细胞后7 - 14天，肿瘤内β-半乳糖苷酶染色最强。收获的肿瘤中的体内转导率在10%至70%之间。没有证据表明周围正常神经组织发生转导。偶尔观察到肿瘤内或肿瘤附近的血管内皮细胞呈5-溴-4-氯-3-吲哚-β-D-吡喃半乳糖苷阳性。（摘要截短至400字）
