Gene Therapy with SLPI Promoter Controlled Replication-competent Adenovirus for Non-small Cell Lung Cancer

分泌性白细胞蛋白酶抑制剂（SLPI）在几乎所有非小细胞肺癌（NSCLC）中高表达，但在大多数其他肿瘤类型中不表达。为了建立针对非小细胞肺癌的特异性基因疗法，我们构建了AdSLPIE1AdB，这是一种腺病毒载体，具有双表达盒，由SLPI启动子基因驱动的E1A和在CMV启动子控制下的E1B-19K组成，只能在非小细胞肺癌细胞中选择性复制。AdSLPI感染。E1AdB产生E1A蛋白表达和腺病毒复制，仅在产生slpi的NSCLC细胞（A549、H358和HS24）中导致病毒滴度增加100倍以上。相反，在不产生slpi的HepG2细胞中，既没有检测到E1A蛋白，也没有检测到复制。AdSLPI治疗。EIAdB在体外以剂量依赖性方式显著抑制NSCLC细胞的增殖，而AdSLPI对HepG2或正常人支气管上皮细胞的细胞生长没有影响。E1AdB感染。AdSLPI直接注射。与对照组相比，E1AdB注入裸小鼠A549和H358肿瘤可显著降低肿瘤生长（A549: 57%, p<0.02, H358: 67%, p<0.03）。组织学检查显示AdSLPI复制。E1AdB和强诱导坏死和凋亡。此外，我们评估了AdSLPI的组合。E1AdB和AdCMVNK4编码具有较强抗血管生成活性的NK4蛋白E1A由AdSLPI表达。E1AdB反式作用于AdCMVNK4的复制，从而增加NK4的表达。将这两种载体注射到H358肿瘤中，与单独注射每种载体相比，肿瘤生长的降低更为显著。这些结果表明AdSLPI。E1AdB可为非小细胞肺癌提供一种选择性的治疗方式，并且与AdSLPI联合使用。E1AdB和AdCMVNK4可能是治疗非小细胞肺癌更有效的基因疗法。

Secretory leukoprotease inhibitor (SLPI) is highly expressed in almost all non-small cell lung cancers (NSCLC), but not in the majority of other tumor types. In an attempt to create a specific gene therapy for NSCLC, we constructed AdSLPIE1AdB, an adenovirus vector with a double expression cassette consisting of E1A driven by the SLPI promoter gene followed by E1B-19K under the control of the CMV promoter that can selectively replicate only in NSCLC cells. Infection of AdSLPI.E1AdB yielded E1A protein expression and adenovirus replication resulting in a more than 100 fold increase of the virus titers only in SLPI-producing NSCLC cells (A549, H358, and HS24). In contrast, neither E1A protein nor replication was detected in non SLPI-producing HepG2 cells. Treatment with AdSLPI.EIAdB significantly inhibited the proliferation of NSCLC cells in vitro in a dose dependent manner, whereas the cell growth of HepG2 or normal human bronchial epithelial cells was not affected by AdSLPI.E1AdB infection. Direct injection of AdSLPI.E1AdB into A549 and H358 tumors in nude mice resulted in a marked reduction in tumor growth compared to controls (A549: 57%, p<0.02, H358: 67%, p<0.03). Histological examination revealed the replication of AdSLPI.E1AdB and strong induction of necrosis and apoptosis. In addition, we evaluated the combination of AdSLPI.E1AdB and AdCMVNK4 encoding NK4 protein which has strong anti-angiogenic activity E1A expressed by AdSLPI.E1AdB trans-acts on the replication of AdCMVNK4 and thus increases the expression of NK4. Injection of these two vectors into H358 tumors resulted i n a more striking reduction of tumor growth compare to single injection of each vector. These results suggest that AdSLPI.E1AdB could provide a selective therapeutic modality for NSCLC and that the combination of AdSLPI.E1AdB and AdCMVNK4 maybe a more effective gene therapy for NSCLC.