Buñuales, M. (María)
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- Enhanced therapeutic effect using sequential administration of antigenically distinct oncolytic viruses expressing oncostatin M in a Syrian hamster orthotopic pancreatic cancer model(BioMed Central, 2015) González-Aseguinolaza, G. (Gloria); Bravo-Perez, C. (Carlos); Quetglas, J.I. (José Ignacio); Larrea, E. (Esther); Poutou, J. (Joanna); Nistal-Villan, E. (Estanislao); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén); Buñuales, M. (María); Carte, B. (Beatriz); Gonzalez-Aparicio, M. (Manuela)The limited efficacy of current treatments against pancreatic cancer has prompted the search of new alternatives such as virotherapy. Activation of the immune response against cancer cells is emerging as one of the main mechanisms of action of oncolytic viruses (OV). Direct oncolysis releases tumor antigens, and viral replication within the tumor microenvironment is a potent danger signal. Arming OV with immunostimulatory transgenes further enhances their therapeutic effect. However, standard virotherapy protocols do not take full advantage of OV as cancer vaccines because repeated viral administrations may polarize immune responses against strong viral antigens, and the rapid onset of neutralizing antibodies limits the efficacy of redosing. An alternative paradigm based on sequential combination of antigenically distinct OV has been recently proposed.
- Deletion of the E3-6.7K/gp19K region reduces the persistence of wild-type adenovirus in a permissive tumor model in Syrian hamsters(Nature, 2009) Lamas, O. (Óscar); Aldabe, R. (Rafael); Alzuguren, P. (Pilar); Prieto, J. (Jesús); Bortolanza, S. (Sergia); Hernandez-Alcoceba, R. (Rubén); Buñuales, M. (María)
- Short-term local expression of a PD-L1 blocking antibody from a self-replicating RNA vector induces potent antitumor responses(2019) Kochan, G. (Grazyna); Mancheño, U. (Uxua); Sanchez-Paulete, A.R. (Alfonso R.); Smerdou, C. (Cristian); Galindo, J. (Javier); Ballesteros-Briones, M.C. (María Cristina); Hervas-Stubbs, S. (Sandra); Melero, I. (Ignacio); Casales, E. (Erkuden); Prieto, J. (Jesús); Martisova, E. (Eva); Gorraiz, M. (Marta); Escors, D. (David); Hernandez-Alcoceba, R. (Rubén); Buñuales, M. (María); Silva-Pilipich, N.R. (Noelia Romina); Lasarte, J.J. (Juan José)Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFVaPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and s
- Evaluation of monocytes as carriers for armed oncolytic adenoviruses in murine and Syrian hamster models of cancer(Mary Ann Liebert, 2012) Garcia-Aragoncillo, E. (Eva); Quetglas, J.I. (José Ignacio); Ortiz-de-Solorzano, C. (Carlos); Hervas-Stubbs, S. (Sandra); Prieto, J. (Jesús); Bortolanza, S. (Sergia); Hernandez-Alcoceba, R. (Rubén); Benavides, C. (Carolina); Buñuales, M. (María); RaquelReplication-competent (oncolytic) adenoviruses (OAV) can be adapted as vectors for the delivery of therapeutic genes, with the aim of extending the antitumor effect beyond direct cytolysis. Transgene expression using these vectors is usually intense but short-lived, and repeated administrations are hampered by the rapid appearance of neutralizing antibodies (NAbs). We have studied the performance of monocytes as cell carriers to improve transgene expression in cancer models established in athymic mice and immunocompetent Syrian hamsters. Human and hamster monocytic cell lines (MonoMac6 and HM-1, respectively) were loaded with replication-competent adenovirus-expressing luciferase. Intravenous administration of these cells caused a modest increase in transgene expression in tumor xenografts, but this effect was virtually lost in hamsters. In contrast, intratumoral administration of HM-1 cells allowed repeated cycles of expression and achieved partial protection from NAbs in preimmunized hamsters bearing pancreatic tumors. To explore the therapeutic potential of this approach, HM-1 cells were loaded with a hypoxia-inducible OAV expressing the immunostimulatory cytokine interleukin-12 (IL-12). Three cycles of treatment achieved a significant antitumor effect in the hamster model, and transgene expression was detected following each administration, in contrast with the rapid neutralization of the free virus. We propose monocytes as carriers for multiple intratumoral administrations of armed OAVs.
- Epilepsy and neuropsychiatric comorbidities in mice carrying a recurrent Dravet syndrome SCN1A missense mutation(Springer Science and Business Media LLC, 2019) Mora-Jiménez, L. (Lucía); Artieda, J. (Julio); Nicolas, M.J. (María Jesús); Puerta, E. (Elena); Ricobaraza, A. (Ana); Mingorance, A. (Ana); Sanchez-Carpintero, R. (Rocío); Valencia, M. (Miguel); Besne, G. (Guillermo); Sola-Sevilla, N. (Noemi); Hernandez-Alcoceba, R. (Rubén); Buñuales, M. (María); Gonzalez-Aparicio, M. (Manuela)Dravet Syndrome (DS) is an encephalopathy with epilepsy associated with multiple neuropsychiatric comorbidities. In up to 90% of cases, it is caused by functional happloinsufficiency of the SCN1A gene, which encodes the alpha subunit of a voltage-dependent sodium channel (Nav1.1). Preclinical development of new targeted therapies requires accessible animal models which recapitulate the disease at the genetic and clinical levels. Here we describe that a C57BL/6 J knock-in mouse strain carrying a heterozygous, clinically relevant SCN1A mutation (A1783V) presents a full spectrum of DS manifestations. This includes 70% mortality rate during the first 8 weeks of age, reduced threshold for heat-induced seizures (4.7 °C lower compared with control littermates), cognitive impairment, motor disturbances, anxiety, hyperactive behavior and defects in the interaction with the environment. In contrast, sociability was relatively preserved. Electrophysiological studies showed spontaneous interictal epileptiform discharges, which increased in a temperature-dependent manner. Seizures were multifocal, with different origins within and across individuals. They showed intra/inter-hemispheric propagation and often resulted in generalized tonic-clonic seizures. 18F-labelled flourodeoxyglucose positron emission tomography (FDG-PET) revealed a global increase in glucose uptake in the brain of Scn1aWT/A1783V mice. We conclude that the Scn1aWT/A1783V model is a robust research platform for the evaluation of new therapies against DS.
- Application of a split-Cre system for high-capacity adenoviral vector amplification(Wiley, 2023) Prieto, J. (Jesús); Ortiz-de-Landazuri, I. (Iñaki); Hernandez-Alcoceba, R. (Rubén); Buñuales, M. (María); Gonzalez-Aparicio, M. (Manuela)Background and aims: High-capacity adenoviral vectors (HC-AdV) show extended DNA payload and stability of gene expression in vivo due to the absence of viral coding sequences. However, production requires methods to trans-complement viral proteins, usually through Helper Viruses (HV). The Cre/loxP system is frequently employed to remove the packaging signal in HV genomes, in order to avoid their encapsidation. However, chronic exposure to the Cre recombinase in packaging cells is detrimental. We have applied the dimerizable Cre system to overcome this limitation. Methods and results: Cre was split in two fragments devoid of recombinase function (N-terminal 244 and C-terminal 99 amino-acids). In one version of the system, interaction with both moieties was favored by rapamycin-dependent heterodimerization domains (DiCre). Other version contained only Cre sequences (oCre). We generated packaging cells and HVs expressing the complementary fragments and studied their performance for HC-AdV production. We found that both conformations avoided interference with the growth of packaging cells, and the oCre system was particularly suitable for HC-AdV amplification. Conclusions: The split-Cre system improves the performance of packaging cells and can reduce the time and cost of HC-AdV amplification up to 30% and 15%, respectively. This may contribute to the standardization of HC-AdV production.
- Human adenovirus replicates in immunocompetent models of pancreatic cancer in syrian hamsters(Mary Ann Liebert, 2007) Qian, C. (Cheng); Alzuguren, P. (Pilar); Prieto, J. (Jesús); Bortolanza, S. (Sergia); Hernandez-Alcoceba, R. (Rubén); Buñuales, M. (María)
- Preferential expression of SCN1A in GABAergic neurons improves survival and epileptic phenotype in a mouse model of Dravet syndrome(2023) Rubinstein, M. (Moran); Honrubia, A. (Adriana); Vides-Urrestarazu, I. (Irene); Ricobaraza, A. (Ana); Lanciego, J.L. (José Luis); Fadila, S. (Saja); Sanchez-Carpintero, R. (Rocío); Roda, E. (Elvira); Arnaiz, P. (Patricia); Banderas, J. (Julliana); Sola-Sevilla, N. (Noemi); Hernandez-Alcoceba, R. (Rubén); Buñuales, M. (María); Gonzalez-Aparicio, M. (Manuela)The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease. KEY MESSAGES: Adenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting. An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons. A hybrid promoter allows preferential expression of transgenes in GABAergic neurons. Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model.
- Transfer of SCN1A to the brain of adolescent mouse model of Dravet syndrome improves epileptic, motor, and behavioral manifestations(Elsevier, 2021) Rubinstein, M. (Moran); González-Aseguinolaza, G. (Gloria); Mora-Jiménez, L. (Lucía); Nicolas, M.J. (María Jesús); Miguelez, C. (Cristina); Puerta, E. (Elena); Ricobaraza, A. (Ana); Fadila, S. (Saja); Sanchez-Carpintero, R. (Rocío); Valencia, M. (Miguel); Lumbreras, S. (Sara); Tønnesen, J. (Jan); Hernandez-Alcoceba, R. (Rubén); Buñuales, M. (María); Gonzalez-Aparicio, M. (Manuela); Fernandez-Pierola, E. (Eva); Gimenez-Minguez, P. (Paula)Dravet syndrome is a genetic encephalopathy characterized by severe epilepsy combined with motor, cognitive, and behavioral abnormalities. Current antiepileptic drugs achieve only partial control of seizures and provide little benefit on the patient’s neurological development. In >80% of cases, the disease is caused by haploinsufficiency of the SCN1A gene, which encodes the alpha subunit of the Nav1.1 voltage-gated sodium channel. Novel therapies aim to restore SCN1A expression in order to address all disease manifestations. We provide evidence that a high-capacity adenoviral vector harboring the 6-kb SCN1A cDNA is feasible and able to express functional Nav1.1 in neurons. In vivo, the best biodistribution was observed after intracerebral injection in basal ganglia, cerebellum, and prefrontal cortex. SCN1A A1783V knockin mice received the vector at 5 weeks of age, when most neurological alterations were present. Animals were protected from sudden death, and the epileptic phenotype was attenuated. Improvement of motor performance and interaction with the environment was observed. In contrast, hyperactivity persisted, and the impact on cognitive tests was variable (success in novel object recognition and failure in Morris water maze tests). These results provide proof of concept for gene supplementation in Dravet syndrome and indicate new directions for improvement.
- Treatment of pancreatic cancer with an oncolytic adenovirus expressing Interleukin-12 in syrian hamsters(Nature Publishing Group, 2009) González-Aseguinolaza, G. (Gloria); Ortiz-de-Solorzano, C. (Carlos); Otano, I. (Itziar); Prieto, J. (Jesús); Bortolanza, S. (Sergia); Hernandez-Alcoceba, R. (Rubén); Buñuales, M. (María); Perez-Martin, D. (Daniel)