使用腫瘤內電衝法轉染人類IL-12基因治療犬傳染性花柳性腫瘤

Abstract

在近幾年的文獻指出，IL-12基因治療可以引起腫瘤消退，抑制遠端轉移，產生抗腫瘤反應，不過都是在實驗小鼠模式，無文獻報告IL-12在較大型動物的抗腫瘤效果，基因治療在小鼠模式的結果並不總是適用於人類，較大型動物的臨床前模式對於人類應用是較有意義的，因為較大型動物的生物特性與人類相近，例如實驗犬的體重與器官大小與人類差異較小，以實驗犬的研究結果，在人類臨床反應上的差異較接近，對於人類腫瘤的基因治療是很適當的臨床前模式。因此本篇研究以構築IL-12基因的質體，利用腫瘤內電衝方式治療犬傳染性花柳性腫瘤，報告此基因治療的效果與抗腫瘤機制。首先，我們發現在CTVT模式中，最適當的電衝條件如下：電壓為200 V、 電衝10次、每次持續50毫秒且使用2針之針狀電極進行基因治療。此外我們更進一步證實，電衝本身並不會影響腫瘤的生長；另外當phIL-12質量固定，腫瘤的治療效果與電衝次數無關。完成以上基因治療前的測試後，我們利用構築人類IL-12基因的質體，以腫瘤內電衝治療CTVT。結果顯示進行治療後，可以有效抑制腫瘤生長，使腫瘤完全消退或呈現液化狀態，並且持續產生抗腫瘤的保護力，對遠端已發展但未治療腫瘤的生長產生抑制效果；對再次接種的腫瘤也能抑制其生長。電衝後腫瘤與血液中IL-12的濃度增加，在腫瘤內也測得IFN-γ的RNA產生。同時發現腫瘤內浸潤淋巴球增加，包括CD4+T和CD8+T細胞。除此之外腫瘤細胞的MHC大為增加，大量的腫瘤細胞產生凋亡；此外我們發現以0.3mg之IL-12質體治療的效果與1mg之IL-12質體相近；在整個研究過程中，實驗犬並無任何毒性產生。以上這些結果顯示使用腫瘤內電衝，進行IL-12基因治療，在實驗犬模式，治療CTVT的效果是非常明顯，期望對於人類臨床之腫瘤治療是有所助益。

Interleukin-12（IL-12）has recently been demonstrated that local or systemic treatment mediates profound antitumor effects, causing regression of established tumors and their distant metastases in small laboratory animals. But, there is lack of data that IL-12 on tumor effect in larger animal models than mice. Gene therapy studies performed with small-inbred laboratory animals are not always as transferable to humans as investigators would like. Larger animal preclinical models have the potential of being useful intermediate steps between rodent studies and human applications. Biological similarities such as body weight and organ size between dogs and humans make the dog an appropriate preclinical model for gene therapy of human tumors. Therefore, we reported here the effectiveness of gene therapy with plasmid encoded IL-12 through in vivo electroporation to treat canine transmissible venereal tumor (CTVT). The optimal conditions of electroporation for gene transfer into CTVT by luciferase activity were as follows: voltage at 200 V; duration at 50 ms; the number of shocks at 10 pulses and electrode with 2 needles. Electroporation only without IL-12 gene did not affect the growth of CTVT. Different injection frequencies with same amount of IL-12 plasmid made no difference for the growth of CTVT. Intratumoral administration of the phIL-12 significantly inhibited the growth of CTVT and suppressed the growth of distant CTVT. This treatment elevated IL-12 in tumors and serum. The expression of IFN-γ RNA was also observed in these tumors. In addition, the IL-12 gene therapy induced more infiltrating lymphocytes in the tumors. The lymphocytes were identified as CD4+ and CD8+T cells. The surface expression of MHC molecules on CTVT cells was significantly increased after gene therapy. More apoptotic tumor cells were also found. The inhibition of tumor growth was similar with either dosage of 0.3mg and 1 mg IL-12 plasmid delivered by electroporation. The treatment did not result in any toxicity in the dogs. We concluded that gene therapy with electroporation to treat CTVT in vivo using IL-12 is very efficient and is thus promising for further clinical trial.