Use of a Novel Integrase-Deficient Lentivirus for Targeted Anti-Cancer Therapy With Survivin Promoter-Driven Diphtheria Toxin A

Abstract

As an immunotoxin, diphtheria toxin has been widely used in gene therapy and gene function assays for its roles in protein synthesis inhibition, and the aim of our study is to set up a nonintegrating lentiviral system for specific expression of diphtheria toxin A (DTA) used in cancer gene therapy.Here, we established a lentiviral system that could coordinately express fluorescent protein and DTA driven by the cytomegalovirus (CMV) promoter, which is convenient for us to precisely trace the expression of DTA and monitor the process of lentivirus packaging. To achieve safer cancer therapy, we replaced the CMV promoter with the Survivin promoter, a specific promoter that is dramatically activated in cancer tissues and cells, but not in normal tissues and cells, and that will impose greater therapeutic potential because a significant expression difference occurred between these 2 groups. Meanwhile, we obtained integrase-deficient lentivirus (IDLV) after packaging with the integrase mutant, which expresses defective integrase RRK262263264AAH, to minimize the side effects that derived from the insertional mutagenesis of the host genome.Our results suggest that the IDLV system that we generated possesses therapeutic potential in cancers in vitro and in vivo.

FIGURE 1

Survivin protein and mRNA levels are dramatically increased in cancer tissues. 1, normal rectum tissue; 2, esophageal squamous cell carcinoma adjacent tissue; 3, esophageal squamous cell carcinoma tissue; 4, gastric cancer adjacent tissue; 5, gastric cancer tissue; 6, breast cancer tissue; 7, rectal cancer adjacent tissue; 8, rectal cancer tissue. (A) Comparison of the protein levels in normal and cancer tissues by western blotting. The protein levels of Survivin are normalized with the β-actin protein level. The analysis and histogram of the protein level are described above the protein bands; (B) Analysis of the mRNA levels in normal and cancer tissues by quantitative polymerized chain reaction. The mRNA levels of Survivin are normalized with the β-actin mRNA level.

FIGURE 2

Survivin protein and mRNA levels are dramatically increased in cancer cells. (A) Comparison of the protein levels in normal and cancer cells by western blotting. The protein levels of Survivin are normalized with the β-actin protein level. The analysis and histogram of the protein level are described above the protein bands; (B) Analysis of mRNA levels in normal and cancer cells by quantitative polymerized chain reaction. The mRNA levels of Survivin are normalized with the β-actin mRNA level.

FIGURE 3

Clone different sizes of the Survivin promoter and compare their activities in cancer cells. (A) Clone of Survivin promoters and construction of pGL3 reporter vectors as driven by the Survivin promoter. Lane 1 is the PCR product of Surp269, lane 2 is the product of pGL3-Surp269-Luciferase digested with the KpnI and NheI enzymes, lane 3 is the PCR product of Surp1430, and lane 4 is the product of pGL3-Surp1430-Luciferase digested with KpnI and NheI enzymes; (B) Diagram of the Survivin promoter-Luc construct with the firefly luciferase gene under the control of Surp1430 or Surp269; (C) Comparison of the Surp1430 and Surp269 activities in different cancer cells. PCR = polymerized chain reaction.

FIGURE 4

In vitro analyses of the Surp1430 and CMV promoter activities through transient transfection. The transcriptional activities of the Surp1430 and CMV promoters were measured in normal and multiple cancer cells. CMV = cytomegalovirus.

FIGURE 5

In vitro assays of killing effect on cancer cells with DTA-expressing lentiviral vectors driven by Surp1430 promoters. (A) Lentiviral vectors comprising the coordinate expression of fluorescent protein and DTA as driven by Surp1430 specifically kill cancer cells in vitro. Cell proliferation was inhibited and cell counts decreased after the transient transfection of DTA-expressing lentiviral vectors as driven by Surp1430 in cancer cells. The 0-μg group represents the co-transfection of 0.3 μg of pPRIME-CMV-GFP-FF3, 0.2 μg of pGL3-control and 25 ng of pRL-TK into cells; the 0.1-μg group represents the co-transfection of 0.1 μg of pPRIME-Surp1430-GFP-2A-DTA-FF3, 0.2 μg of pPRIME-CMV-GFP-FF3, 0.2 μg of pGL3-control and 25 ng of pRL-TK into cells; and the 0.3-μg group represents the co-transfection of 0.3 μg of pPRIME-Surp1430-GFP-2A-DTA-FF3, 0.2 μg of pGL3-control and 25 ng of pRL-TK into cells; (B) Quantifying the RNA level of DTA in multiple cells after transfection with DTA-expressing lentiviral vectors. CMV = cytomegalovirus, DTA = diphtheria toxin A.

FIGURE 6

Production of IDLVs after the co-transfection of lentiviral plasmids and packaging plasmids into HEK293T[mEF-2(G717R)] cells. (A) Production of pPRIME-CMV-GFP-FF3 IDLVs in cells; (B) Production of pPRIME-CMV-GFP-2A-DTA-FF3 IDLVs in cells; (C) Production of pPRIME-Surp1430-GFP-FF3 IDLVs in cells; (D) Production of pPRIME-Surp1430-GFP-2A-DTA-FF3 IDLVs in cells. CMV = cytomegalovirus, IDLV = integrase-deficient lentivirus.

FIGURE 7

The effect of the intratumoral injection of DTA-expressing IDLVs on subcutaneous breast tumor growth in nude mice (n = 5). (A) The tumor growth curve after the repeated injection of pPRIME-CMV-GFP-FF3, pPRIME-Surp1430-GFP-FF3 IDLVs, pPRIME-CMV-GFP-2A-DTA-FF3, and pPRIME-Surp1430-GFP-FF3 IDLVs; (B) The dissection of tumors from nude mice after multiple injection of IDLVs, the injected IDLVs are described above the tumors; (C) The rate of increased body weight between mice after injection with IDLVs. Before the initial injection, the whole body weight of mice was recorded, the value of body weight was also recorded after tumors dissection, and the increased value was calculated, then the effect of IDLVs on body weight change was evaluated when compared with group injected with pPRIME-CMV-GFP-FF3. DTA = diphtheria toxin A; IDLV = integrase-deficient lentivirus. CMV, cytomegalovirus.