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）在几乎所有的非小细胞肺癌（NSCLC）中高度表达，但在大多数其他肿瘤类型中不表达。为了建立一种针对NSCLC的特异性基因治疗方法，我们构建了AdSLPIE 1AdB，一种具有双表达盒的腺病毒载体，该双表达盒由SLPI启动子基因驱动的E1 A和CMV启动子控制的E1 B-19 K组成，可以选择性地仅在NSCLC细胞中复制。AdSLPI.E1AdB的感染产生E1 A蛋白表达和腺病毒复制，导致仅在产生SLPI的NSCLC细胞（A549、H358和HS 24）中病毒滴度增加超过100倍。相反，在非SLPI产生的HepG 2细胞中既没有检测到E1 A蛋白也没有检测到复制。AdSLPI. E1 AdB能明显抑制NSCLC细胞的增殖，且呈剂量依赖性，而对HepG 2细胞和正常人支气管上皮细胞的生长无明显影响。将AdSLPI.E1AdB直接注射到裸鼠中的A549和H358肿瘤中导致与对照相比肿瘤生长显著减少（A549：57%，p<0.02，H358：67%，p<0.03）。组织学检查显示AdSLPI.E1AdB复制并强烈诱导坏死和凋亡。此外，我们评估了AdSLPI.E1AdB和编码NK 4蛋白的AdCMVNK 4的组合，其具有强的抗血管生成活性，AdSLPI.E1AdB表达的E1 A反式作用于AdCMVNK 4的复制，从而增加NK 4的表达。将这两种载体注射到H358肿瘤中与单次注射每种载体相比导致肿瘤生长更显著的减少。结论AdSLPI. E1 AdB可为NSCLC提供一种选择性的治疗模式，联合AdCMVNK 4基因治疗NSCLC可能更为有效。

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.