Saporin as a novel suicide gene in anticancer gene therapy

Zarovni, Nataša; Vago, Riccardo; Soldà, T; Monaco, Lucía; Fabbrini, Maria Serena

doi:10.1038/sj.cgt.7700998

Cited by 34 publications

(28 citation statements)

References 48 publications

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“…In order to study the usage of RTA as a reporter, it was chemically conjugated to an enzymatically inactive variant of saporin (saporin-KQ), previously reported in the literature [17]. Saporin-KQ was cloned into an expression vector, and the presence of the insert was confirmed by DNA sequencing.…”

Section: Resultsmentioning

confidence: 99%

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Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics

Gilabert-Oriol

Thakur

Mallinckrodt

et al. 2014

Toxins

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Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters—horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)—were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates—saporin-HRP, Alexasaporin and saporin-KQ-RTA—were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of Alexasaporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10–1000 nM.

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Section: Resultsmentioning

confidence: 99%

“…In case of ricin A-chain as a reporter, cytotoxicity is the readout of the experiment. Therefore, a previously described enzymatically inactive variant of saporin (saporin-KQ) [17] was used as a protein that escapes from endo/lysosomes in the presence of triterpenoidal saponins, but that lacks cytotoxic properties. …”

Section: Introductionmentioning

confidence: 99%

Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics

Gilabert-Oriol

Thakur

Mallinckrodt

et al. 2014

Toxins

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“…This approach to gene delivery through targeting of the TfR has been termed ''transferrinfection'' (64). The saporin gene has already been shown to be a suitable candidate gene for this purpose (65). Specificity could be further enhanced by delivery of the saporin gene under the control of a cell-specific promoter, such as the immunoglobulin promoter to target malignant B cells.…”

Section: Discussionmentioning

confidence: 99%

Conjugation of an anti–transferrin receptor IgG3-avidin fusion protein with biotinylated saporin results in significant enhancement of its cytotoxicity against malignant hematopoietic cells

Daniels¹,

Delgado³

et al. 2007

Molecular Cancer Therapeutics

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We have previously developed an antibody fusion protein composed of a mouse/human chimeric IgG3 specific for the human transferrin receptor genetically fused to avidin (anti-hTfR IgG3-Av) as a universal delivery system for cancer therapy. This fusion protein efficiently delivers biotinylated FITC into cancer cells via TfR-mediated endocytosis. In addition, anti-hTfR IgG3-Av alone exhibits intrinsic cytotoxic activity and interferes with hTfR recycling, leading to the rapid degradation of the TfR and lethal iron deprivation in certain malignant B-cell lines. We now report on the cytotoxic effects of a conjugate composed of anti-hTfR IgG3-Av and biotinylated saporin 6 (b-SO6), a toxin derived from the plant Saponaria officinalis that inhibits protein synthesis. Conjugation of anti-hTfR IgG3-Av with b-SO6 enhances the cytotoxic effect of the fusion protein in sensitive cells and also overcomes the resistance of malignant cells that show low sensitivity to the fusion protein alone. Our results show for the first time that loading anti-hTfR IgG3-Av with a biotinylated toxin enhances the cytotoxicity of the fusion protein alone. These results suggest that anti-hTfR IgG3-Av has great potential as a therapeutic agent for a wide range of applications due to its intrinsic cytotoxic activity plus its ability to deliver biotinylated molecules into cancer cells.

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“…Examples of cell death genes are endonucleases such as DNase I, or a cytotoxin such as saporin. Saporin is a ribosome-inactivating protein from seeds of the plant Saponaria officinalis, and a study [55] has demonstrated the efficacy of using a saporin gene in cancer gene therapy via a non-viral gene delivery approach. The HPV18-LCR800 luciferase promoter construct had approximately 17-fold lower activity than an SV40 (early promoter) pGL3 luciferase vector (average of three experiments) (data not shown) and this is in line with other studies [56].…”

Section: Resultsmentioning

confidence: 99%

The use of a human papillomavirus 18 promoter for tissue-specific expression in cervical carcinoma cells

Lung

Mak

Murray

2011

Cellular and Molecular Biology Letters

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Abstract:The use of tissue-specific promoter elements in the treatment of cervical cancer has been explored in this paper. The P 105 promoter of human papillomavirus 18 (HPV18) was utilised to direct tissue-specific expression in a number of cell types. Expression was examined in three cervical carcinoma cell lines: HeLa (HPV18 positive), SiHa (HPV16 positive), and C33A cells (HPV negative); the epithelial cell line, H1299; and the foetal fibroblast cell line, MRC5, utilising a luciferase expression vector. Expression was highest in the cervical cell lines by a factor of at least 80. The effect of a number of mutations in the P 105 promoter on expression levels was examined. Three deletion constructs of the long control region (LCR) were investigated: an 800 bp fragment (LCR800), a 400 bp fragment (LCR400), and a 200 bp fragment (LCR200), as well as the full length product LCR of HPV18 (LCR1000). The LCR800 construct of the HPV18 P 105 promoter had the highest level of expression in the cervical cell lines and was also highest in the HPV18-positive HeLa cell line. Site-directed mutagenesis was then employed on the LCR800 construct to create four further constructs that each had inactivating mutations in one of the four E2 binding sites (E2BSs). Overall, this study indicated that the LCR800 construct of the HPV18 P 105 promoter could be utilised as a tissuerestricted promoter in cervical cancer cells.

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Saporin as a novel suicide gene in anticancer gene therapy

Cited by 34 publications

References 48 publications

Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics

Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics

Conjugation of an anti–transferrin receptor IgG3-avidin fusion protein with biotinylated saporin results in significant enhancement of its cytotoxicity against malignant hematopoietic cells

The use of a human papillomavirus 18 promoter for tissue-specific expression in cervical carcinoma cells

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