Desarrollo de un sistema de expresión inducible y hepato-específico para virus adeno-asociados y su aplicación en la inmunoterapia de metástasis hepáticas de carcinoma colorrectal
- Autores: Lucía María Vanrell Majo
- Directores de la Tesis: Gloria González Aseguinolaza (dir. tes.)
- Lectura: En la Universidad de Navarra ( España ) en 2009
- Idioma: español
- Tribunal Calificador de la Tesis: Miguel Chillón Rodríguez (presid.), Rubén Hernández-Alcoceba (secret.), Cristian Smerdou Picazo (voc.), Cristina Fillat Fonts (voc.), Juan Carlos Ramírez Martínez (voc.)
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- Resumen
DEVELOPMENT OF AN INDUCIBLE AND LIVER-SPECIFIC ADENO-ASSOCIATED VECTOR AND ITS APPLICATION ON COLORECTAL CANCER LIVER METASTASES IMMUNOTHERAPY.
Luc¿ªa Vanrell Maj¿®, Faculty of Science, University of Navarra (Spain), 2009.
Interleukin-12 (IL12) is a multifunctional cytokine that stimulates both innate and adaptive immunity, acting as a key regulator of cell-mediated immune responses. The immunomodulating and antiangiogenic functions of IL12 have provided the rationale for exploiting this cytokine as an anticancer agent. The promising data obtained by the administration of IL12 recombinant protein in preclinical animal models of cancer raised hopes that recombinant IL12 could be a powerful therapeutic agent. However, clinical trials revealed a modest clinical response that was limited by the development of severe toxicity when high doses of this cytokine were used. Gene therapy can significantly increase cytokine expression in the target organ without excessively elevating systemic cytokine levels, which leads to an increased efficacy/toxicity ratio. Early clinical trials with short-term IL12 expression vectors have set the proof-of-concept that local production of IL12 inside a tumor can stimulate tumor infiltration by effector immune cells, sometimes followed by tumor regression. New vectors that might achieve regulated, long-term production of this cytokine might have better results and merit clinical testing. Recombinant adeno-associated viral (AAV) vectors have unique properties, which make them suitable vectors for long-term gene transfer. The aim or this work was to develop liver-specific and inducible adeno-associated vectors delivering IL12 for the treatment of liver metastases of colorectal carcinoma.
First of all, we assessed the liver transduction efficiency and biodistribution of AAV-pseudotyped capsids (serotypes) 1, 5, 6, and 8, combined with single-stranded and double-stranded genomic AAV2 structures carrying the luciferase reporter gene after systemic administration. The analysis was performed in vivo and ex vivo, in male and female mice. Gender-related differences in AAV-mediated transduction and biodistribution were shown for the four serotypes. Our data confirm the superiority of AAV8 over the rest of the serotypes, as well as a significant advantage of double-stranded genomes in terms of liver transduction efficiency, particularly in females.
Once we have chosen AAV serotype 8, we designed a bidirectional and autoregulatory TetON system in which transcription of both, the reverse tetracycline transactivator (M2) and the transgene is initiated from a bidirectional tetracyline-responsive promoter and terminated at bidirectional SV40 polyadenilation sites, flanking the ITRs. The bidirectional promoter is constituted by 7 tetracycline operator sites flanked by two identical liver-specific promoters (albumin promoters) arranged on opposite directions. We have used the luciferase reporter gene and perform an in vivo characterization of the system. Luciferase activity was highly restricted to the liver as confirmed by ex vivo biodistribution analysis, and high rates of induction were observed in two mice strains (BALB/c and C57BL/6) and both genders. Moreover, this inducible vector showed long term re-induction ability carrying luciferase as the transgene.
Finally, we wanted to analyze the new AAV vector performance on immunotherapy. To that end we used the single chain mouse IL12 transgene, and set up a prevention protocol of colorectal cancer liver metastases, using the C57BL/6-MC38 murine tumor model. The vector induced viral dose-dependent serum levels of mIL12 and mIFN¿Ã, showed no significant toxicity, and was highly efficient in preventing establishment of colorrectal metastasis in the liver. Moreover, it induced an efficient T cell memory response to MC38 cell line.
