Vanrell, L. (Lucía)
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- Development of a liver-specific Tet-on inducible system for AAV vectors and its application in the treatment of liver cancer(Nature, 2011) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Gil-Fariña, I. (Irene); Baldim, V. (Victor); Pañeda, A. (Astrid); Otano, I. (Itziar); Tenenbaum, L. (Lilianne); Beattie, S.G. (Stuart G.); Prieto, J. (Jesús); Vanrell, L. (Lucía); Chtarto, A. (Abdelwahed); Scala, M. (Marianna) Di; Blanco-Fernández, L. (Laura)Recombinant adeno-associated virus (rAAV) are effective gene delivery vehicles that can mediate long-lasting transgene expression. However, tight regulation and tissue-specific transgene expression is required for certain therapeutic applications. For regulatable expression from the liver we designed a hepatospecific bidirectional and autoregulatory tetracycline (Tet)-On system (Tet(bidir)Alb) flanked by AAV inverted terminal repeats (ITRs). We characterized the inducible hepatospecific system in comparison with an inducible ubiquitous expression system (Tet(bidir)CMV) using luciferase (luc). Although the ubiquitous system led to luc expression throughout the mouse, luc expression derived from the hepatospecific system was restricted to the liver. Interestingly, the induction rate of the Tet(bidir)Alb was significantly higher than that of Tet(bidir)CMV, whereas leakage of Tet(bidir)Alb was significantly lower. To evaluate the therapeutic potential of this vector, an AAV-Tet(bidir)-Alb-expressing interleukin-12 (IL-12) was tested in a murine model for hepatic colorectal metastasis. The vector induced dose-dependent levels of IL-12 and interferon-γ (IFN-γ), showing no significant toxicity. AAV-Tet(bidir)-Alb-IL-12 was highly efficient in preventing establishment of metastasis in the liver and induced an efficient T-cell memory response to tumor cells. Thus, we have demonstrated persistent, and inducible in vivo expression of a gene from a liver-specific Tet-On inducible construct delivered via an AAV vector and proved to be an efficient tool for treating liver cancer.
- Long-Term Systemic Expression of a Novel PD-1 Blocking Nanobody from an AAV Vector Provides Antitumor Activity without Toxicity(2020) Smerdou, C. (Cristian); Ballesteros-Briones, M.C. (María Cristina); Hervas-Stubbs, S. (Sandra); Casares, N. (Noelia); Vanrell, L. (Lucía); Martisova, E. (Eva); González-Sapienza, G. (Gualberto); Silva-Pilipich, N.R. (Noelia Romina)Immune checkpoint blockade using monoclonal antibodies (mAbs) able to block programmed death-1 (PD-1)/PD-L1 axis represents a promising treatment for cancer. However, it requires repetitive systemic administration of high mAbs doses, often leading to adverse effects. We generated a novel nanobody against PD-1 (Nb11) able to block PD-1/PD-L1 interaction for both mouse and human molecules. Nb11 was cloned into an adeno-associated virus (AAV) vector downstream of four different promoters (CMV, CAG, EF1α, and SFFV) and its expression was analyzed in cells from rodent (BHK) and human origin (Huh-7). Nb11 was expressed at high levels in vitro reaching 2–20 micrograms/mL with all promoters, except SFFV, which showed lower levels. Nb11 in vivo expression was evaluated in C57BL/6 mice after intravenous administration of AAV8 vectors. Nb11 serum levels increased steadily along time, reaching 1–3 microgram/mL two months post-treatment with the vector having the CAG promoter (AAV-CAG-Nb11), without evidence of toxicity. To test the antitumor potential of this vector, mice that received AAV-CAG-Nb11, or saline as control, were challenged with colon adenocarcinoma cells (MC38). AAV-CAG-Nb11 treatment prevented tumor formation in 30% of mice, significantly increasing survival. These data suggest that continuous expression of immunomodulatory nanobodies from long-term expression vectors could have antitumor effects with low toxicity.
- Adeno-associated viral vectors serotype 8 for cell-specific delivery of therapeutic genes in the central nervous system(Frontiers Media, 2017) Lopez-Franco, E. (Esperanza); González-Aseguinolaza, G. (Gloria); Sucunza, D. (Diego); Vales, A. (África); Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Hommel, M. (Mirja); Dopeso-Reyes, I.G. (Iria G.); Vanrell, L. (Lucía); Pignataro, D. (Diego)Adeno-associated viruses (AAVs) have become highly promising tools for research and clinical applications in the central nervous system (CNS). However, specific delivery of genes to the cell type of interest is essential for the success of gene therapy and therefore a correct selection of the promoter plays a very important role. Here, AAV8 vectors carrying enhanced green fluorescent protein (eGFP) as reporter gene under the transcriptional control of different CNS-specific promoters were used and compared with a strong ubiquitous promoter. Since one of the main limitations of AAV-mediated gene delivery lies in its restricted cloning capacity, we focused our work on small-sized promoters. We tested the transduction efficacy and specificity of each vector after stereotactic injection into the mouse striatum. Three glia-specific AAV vectors were generated using two truncated forms of the human promoter for glial fibrillar acidic protein (GFAP) as well as a truncated form of the murine GFAP promoter. All three vectors resulted in predominantly glial expression; however we also observed eGFP expression in other cell-types such as oligodendrocytes, but never in neurons. In addition, robust and neuron-specific eGFP expression was observed using the minimal promoters for the neural protein BM88 and the neuronal nicotinic receptor β2 (CHRNB2). In summary, we developed a set of AAV vectors designed for specific expression in cells of the CNS using minimal promoters to drive gene expression when the size of the therapeutic gene matters.
- Porphobilinogen deaminase over-expression in hepatocytes, but not in erythrocytes, prevents accumulation of toxic porphyrin precursors in a mouse model of acute intermittent porphyria(Elsevier, 2010) González-Aseguinolaza, G. (Gloria); Dubrot, J. (Juan); Unzu, C. (Carmen); Melero, I. (Ignacio); Prieto, J. (Jesús); Vanrell, L. (Lucía); Enriquez-de-Salamanca, R. (Rafael); Fontanellas-Romá, A. (Antonio); Sampedro, A. (Ana); Mauleon, I. (Itsaso)BACKGROUND & AIMS: Acute intermittent porphyria (AIP) is characterized by hepatic porphobilinogen deaminase (PBGD) deficiency resulting in a marked overproduction of presumably toxic porphyrin precursors. Our study aimed to assess the protective effects of bone marrow transplantation or PBGD gene transfer into the liver against phenotypic manifestations of acute porphyria attack induced in an AIP murine model. METHODS: Lethally irradiated AIP mice were intravenously injected with 5x10(6) nucleated bone marrow cells from wild type or AIP donor mice. To achieve liver gene transfer, AIP mice received via hydrodynamic injection plasmids expressing human PBGD or luciferase, driven by a liver-specific promoter. RESULTS: Erythrocyte PBGD activity increased 2.4-fold in AIP mice receiving bone marrow cells from normal animals. Nevertheless, phenobarbital administration in these mice reproduced key features of acute attacks, such as massively increased urinary porphyrin precursor excretion and decreased motor coordination. Hepatic PBGD activity increased 2.2-fold after hydrodynamic injection of therapeutic plasmid. Mice injected with the luciferase control plasmid showed a high excretion of porphyrin precursors after phenobarbital administration whereas just a small increase was observed in AIP mice injected with the PBGD plasmid. Furthermore, motor disturbance was almost completely abolished in AIP mice treated with the therapeutic plasmid. CONCLUSIONS: PBGD deficiency in erythroid tissue is not associated with phenotypic manifestations of acute porphyria. In contrast, PBGD over-expression in hepatocytes, albeit in a low proportion, reduced precursor accumulation, which is the hallmark of acute porphyric attacks. Liver-directed gene therapy might offer an alternative to liver transplantation applicable in patients with severe and recurrent manifestations.
- Local delivery of optimized nanobodies targeting the PD-1/PD-L1 axis with a self-amplifying RNA viral vector induces potent antitumor responses(Elsevier, 2023) Sarrión, P. (Patricia); Lozano-Moreda, T. (Teresa); Smerdou, C. (Cristian); Igea, A. (Ana); Ballesteros-Briones, M.C. (María Cristina); Vanrell, L. (Lucía); Martisova, E. (Eva); Blanco, E. (E.); Gorraiz, M. (Marta); González-Sapienza, G. (Gualberto); Herrador-Cañete, G. (Guillermo); Silva-Pilipich, N.R. (Noelia Romina); Lasarte, J.J. (Juan José)Despite the success of immune checkpoint blockade for cancer therapy, many patients do not respond adequately. We aimed to improve this therapy by optimizing both the antibodies and their delivery route, using small monodomain antibodies (nanobodies) delivered locally with a self-amplifying RNA (saRNA) vector based on Semliki Forest virus (SFV). We generated nanobodies against PD-1 and PD-L1 able to inhibit both human and mouse interactions. Incorporation of a dimerization domain reduced PD-1/PD-L1 IC50 by 8- and 40-fold for antiPD-L1 and anti-PD-1 nanobodies, respectively. SFV viral particles expressing dimeric nanobodies showed a potent antitumor response in the MC38 model, resulting in >50% complete regressions, and showed better therapeutic efficacy compared to vectors expressing conventional antibodies. These effects were also observed in the B16 melanoma model. Although a short-term expression of nanobodies was observed due to the cytopathic nature of the saRNA vector, it was enough to generate a strong proinflammatory response in tumors, increasing infiltration of NK and CD8+ T cells. Delivery of the SFV vector expressing dimeric nanobodies by local plasmid electroporation, which could be more easily translated to the clinic, also showed a potent antitumor effect.
- IL12-mediated liver inflammation reduces the formation of AAV transcriptionally active forms but has no effect over preexisting AAV transgene expression(Public Library of Science, 2013) González-Aseguinolaza, G. (Gloria); Gil-Fariña, I. (Irene); High, K.A. (Katherine A.); Vales, A. (África); Olagüe, C. (Cristina); Prieto, J. (Jesús); Vanrell, L. (Lucía); Scala, M. (Marianna) Di; Mingozzi, F. (Federico)Recombinant adenoassociated viral vectors (rAAV) have proven to be excellent candidates for gene therapy clinical applications. Recent results showed that cellular immunity to AAV represents a major challenge facing the clinical use of systemic administration of these vectors. Interestingly, no preclinical animal model has previously fully reproduced the clinical findings. The aim of the present work was to enhance the T cell immune response against AAV capsid in mice by the administration of a rAAV expressing the immunostimulatory cytokine IL-12. Our results indicate that although IL-12 expression enhanced the AAV capsid-specific immune response it failed to eliminate transduced hepatocytes and long-term expression was achieved. We found that AAV-mediated transgene expression is altered by IL-12-induced liver inflammation. However, IL-12 expression has no effect over preexisting AAV-mediated transgene expression. IL-12 down-regulates AAV mediated transgene expression via induction of IFN-γ production by NK and T cells, but without altering the transduction efficiency measured by viral genomes. Our results indicate that liver inflammation affects the formation of transcriptionally active AAV vector genomes through an unknown mechanism that can be avoided by the use of DNA-demethylating or anti-inflammatory agents.