Hepatic Intra-arterial Delivery of a “Trojan-horses” Gene Therapy: A Pilot Study on Rabbit VX2 Hepatic Tumor Model

Abstract

Purpose

Gene-directed enzyme prodrug therapy (GDEPT) is a “Trojan-horses” suicide gene therapy that consists of tumor-targeted gene delivery (vectorized by mesenchymal stem cells MSCs) encoding an enzyme that converts a harmless prodrug into cytotoxic metabolites in situ. Then, cytotoxic metabolites passively diffuse in the neighboring tumor cells and kill them (bystander effect). The goal of our study was to assess the feasibility and efficacy of intra-arterial administration of MSCs transduced with an optimized gene (MSC-CYP2B6TM-RED) followed by intravenous administration of cyclophosphamide (CPA) into the VX2 rabbit liver tumor.

Materials and Methods

Nine rabbits with a VX2 liver tumor were randomly assigned into three groups: Control group A (one rabbit) free of any treatment; Control group B (two rabbits) receiving intravenous injection of cyclophosphamide at day 3 and CPA at day 14; and Group C (six rabbits) receiving the GDEPT treatment, consisting of successive intra-arterial injection of transduced-MSCs at days 0 (n = 6) and 11 (n = 3), followed by injection of CPA at days 3 (n = 6) and 14 (n = 3). The tumor response was assessed by ultrasound scan every 7 days and histopathological analysis at sacrifice (D25).

Results

There was a significant difference in the tumor volume between control groups (A + B) and group C at D7: 38/19 cm3 (p = 0.024); D11: 51/20 cm3 (p = 0.024), and D25: 121/37 cm3 (p = 0.048). Tumor necrosis was significantly greater and metastatic spread was lower for rabbits who received GDEPT (78% of total tumor surface) than for control animals (A + B) (22% of total tumor surface (p = 0.006).

Conclusion

Intra-arterial delivery of transduced-MSCs is feasible and, after CPA injection, resulted in 78% tumor necrosis (p = 0.006) and less metastasis in a VX2 liver tumor model.

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Acknowledgements

This work has been selected by the CIRSE 2017 Scientific Programme Committee and the CVIR Editorial Board, and cordially invited to send a complete manuscript for publication in the official journal of CIRSE, the CardioVascular and Interventional Radiology (CVIR). This work was supported by SATT Ile de France and the Ligue Nationale contre le Cancer, comités d’Ile de France. Ikrame Amara was a graduate student funded by the Cancéropole Ile-de-France. Baxter freely provided Cyclophosphamid. We acknowledge Julien Namur, Florentina Pascale from Archimmed SARL for their advice and help in the present work, and Dr. Jean Bénard PharmD, Ph.D, for his comments and editing.

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Correspondence to Olivier Pellerin.

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Conflict of interest

This work was supported by SATT Ile de France and the Ligue Nationale contre le Cancer, comités d’Ile de France. Baxter freely provided Cyclophosphamid. Second author was a graduate student funded by the Cancéropole Ile-de-France. Sixth and seventh authors are the inventor of patent PCT/EP2012/058219, US-2014-0127180A1.

Appendix

Appendix

CYP2B6-TM-RED

As described in previous works [8, 12], we built a CYP2B6TM-RED gene (a fusion of a triple mutant CYP2B6 with NADPH cytochrome P450 reductase) that catalyzed very efficiently the transformation of cyclophosphamide (CPA) into cytotoxic metabolites that causes intra- and inter-strand DNA cross-links and cell death.

Transduced-MSCs

Transduced-MSCs were obtained as previously described by Amara et al. [12]. Briefly, rabbit MSCs from bone-marrow of New Zealand White rabbits (Clinisciences, Nanterre, France) were transduced by lentiviral particles expressing luciferase (Luc2-MSCs). We checked that the pool of rabbit MSCs expressed luciferase at high level by the luminescence assay system (Promega, Madison, Wisconsin); this pool was further used to study MSCs distribution after intra-arterial injection (Fig. 3).

Murine MSCs from the bone-marrow of C57BL/6 mice were provided by Life technologies (GIBCO, Saint Aubin, France) and were tranduced by lentiviral particle LV-CYP2B6TM-RED. After two series of limiting dilutions, the most sensitive clone to CPA (clone 42) [13] was used in in vitro (Fig. 2) and in vivo experiments (Figs. 4, 5, 6).

VX2 Cell Liver Implantation

Tumor carriers were induced by intramuscular injection of 107 VX2 cells (CR2i, APHP-INRA, France) in each hind leg of adult New Zealand White male rabbits. Tumor was allowed to grow for 2 weeks. Then the carrier tumors were excised and minced. Aliquots of fresh tumor chunks (~ 1 mm3) were then implanted in the left hepatic lobe of recipient animals as previously described by Lee et al. [14]. Two weeks following implantation, a 15 cm3 single VX2 liver tumor was identified in the left liver lobe by US scans examination, and treatment was initiated.

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Pellerin, O., Amara, I., Sapoval, M. et al. Hepatic Intra-arterial Delivery of a “Trojan-horses” Gene Therapy: A Pilot Study on Rabbit VX2 Hepatic Tumor Model. Cardiovasc Intervent Radiol 41, 153–162 (2018). https://doi.org/10.1007/s00270-017-1833-8

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Keywords

  • Gene-directed enzyme prodrug therapy
  • Intra-arterial delivery
  • Liver cancer
  • Rabbit VX2