Desarrollo y validación "in vivo" de sistemas de expresión lentiviral regulados por inflamación

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología MolecularLos procesos inflamatorios están presentes en diferentes patologías incluyendo enfermedades inflamatorias crónicas, neuroinflamación y cáncer. Nuestra hipótesis de partida consiste en que un vector lentiviral ideal para el tratamiento con terapia génica puede aprovecharse del nivel de inflamación, específico de cada patología y del curso de la enfermedad, para regular los niveles de expresión del transgén, de forma que se produzcan diferentes niveles de la molécula terapéutica en respuesta al estado inflamatorio local y, por lo tanto, se puedan evitar los efectos secundarios asociados. Nuestro abordaje ha consistido en el desarrollo de vectores virales que contienen promotores inducibles por inflamación para controlar la expresión del transgén. El laboratorio del Dr. Antonio Rodríguez ha desarrollado un nuevo sistema lentiviral inducible por inflamación basado en el promotor de la Selectina-E humana. Además hemos reproducido otros dos sistemas regulados por inflamación previamente descritos: el promotor híbrido formado por el promotor de la IL-6 humana unido a la región potenciadora del promotor humano de la IL-1 y el promotor quimera basado en seis sitios de unión para el factor de transcripción NF-κB en tándem. Hemos validado estos sistemas lentivirales inducibles por inflamación in vitro demostrando su inducibilidad en diferentes tipos celulares (células del sistema inmune, endoteliales, de la glía y tumorales) en respuesta a estímulos inflamatorios como TNF-α, IL-1 y LPS. Se han empleado vectores lentivirales ya que representan la mejor estrategia para obtener una expresión del transgén estable y duradera in vivo. Este estudio demuestra que la combinación de vectores lentivirales con promotores inducibles por inflamación permite la monitorización de procesos inflamatorios como los que tienen lugar en los modelos de inflamación aguda o durante el desarrollo de tumores sólidos. Además nuestros resultados indican que la expresión de citoquinas anti o pro-inflamatorias (IL-10 e IL-12 murinas) bajo el control de promotores regulados por inflamación empleando vectores lentivirales, constituye una estrategia eficiente para conseguir la expresión de la molécula regulada por la propia enfermedad, produciendo un efecto terapéutico en el modelo correspondiente y reduciendo los potenciales efectos secundarios. Estos promotores inducibles pueden representar excelentes sistemas para la expresión regulada por inflamación de diferentes agentes terapéuticos en futuras aplicaciones clínicas.Inflammatory processes are present in different pathologies including chronic inflammatory disorders, neuroinflammatory conditions and cancer. We hypothesized that an ideal lentiviral expression system for gene therapy treatments of such diseases could make use of the pathological inflammation grade, specific to each pathological condition and to the course of the disease, to regulate the transgene expression levels; ideally the expression levels of the therapeutic agent would parallel the local inflammatory status and therefore might avoid undesirable side effects associated with a constant transgene expression. These inflammation-regulated systems might also circumvent drug efficacy attenuation due to a continuous exposure to the therapeutic agent. Our approach has consisted on the generation of lentiviral vectors (LVs) that contain inflammation-regulated promoters to drive transgene expression. Antonio Rodríguez’s research group has developed a novel LV system based on the human E-selectin promoter. In addition we have also generated other two inflammation-regulated LVs based on previous reports: a hybrid promoter based on the human IL-6 promoter plus an enhancer region of the human IL-1 promoter and a chimeric promoter based on six NF-κB transcription factor binding sites in tandem. We have confirmed their in vitro inducibilities in different cell types (immune, endothelial, glia and tumor cells) in response to proinflammatory stimuli such as TNF-α, IL-1 or LPS. LVs represent the chosen gene therapy tool to obtain stable and long-lived transgene expression in vivo. This study shows that the combination of LVs with inflammation inducible promoters allows to monitor inflammatory processes such as those taking place in acute inflammation and solid tumor models. Moreover our results indicate that the expression of the proper cytokine (either murine IL-10 or IL-12) under these inflammation-regulated LV systems constitute an efficient strategy to provide diseaseregulated cytokine expression with therapeutic effects in the mouse model assayed. These systems also reduce side effects as cytokine expression is confined to inflammatory sites. These inducible promoters might represent excellent systems for the inflammation regulated expression of different agents for future clinical applications

IL-10 released by a new inflammation-regulated lentiviral system efficiently attenuates zymosan-induced arthritis

We thank Dr Filip Lim for critical reading of the manuscript, and Dr S. Bartlett for English editing and helpful discussions. We also thank Drs. David Sancho and M. A. del Pozo for providing us with DCs and immortalized MEFs, respectively. AR is supported by Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+I) and Instituto de Salud Carlos III (FIS; PI060122), the Spanish Ministry of Science and Innovation (MICINN;SAF2009-10691) and the Comunidad Autónoma de Madrid (S2006/BIO-0236 and S2010/BMD-2312). JMR is supported by MICINN (RECAVA RD06/0014/005) and by from Fundació La Marató de TV3 (Grant 080731

Genetic interaction between PLK1 and downstream MCPH proteins in the control of centrosome asymmetry and cell fate during neural progenitor division

Alteration of centrosome function and dynamics results in major defects during chromosome segregation and is associated with primary autosomal microcephaly (MCPH). Despite the knowledge accumulated in the last few years, why some centrosomal defects specifically affect neural progenitors is not clear. We describe here that the centrosomal kinase PLK1 controls centrosome asymmetry and cell fate in neural progenitors during development. Gain- or loss-of-function mutations in Plk1, as well as deficiencies in the MCPH genes Cdk5rap2 (MCPH3) and Cep135 (MCPH8), lead to abnormal asymmetry in the centrosomes carrying the mother and daughter centriole in neural progenitors. However, whereas loss of MCPH proteins leads to increased centrosome asymmetry and microcephaly, deficient PLK1 activity results in reduced asymmetry and increased expansion of neural progenitors and cortical growth during mid-gestation. The combination of PLK1 and MCPH mutations results in increased microcephaly accompanied by more aggressive centrosomal and mitotic abnormalities. In addition to highlighting the delicate balance in the level and activity of centrosomal regulators, these data suggest that human PLK1, which maps to 16p12.1, may contribute to the neurodevelopmental defects associated with 16p11.2–p12.2 microdeletions and microduplications in children with developmental delay and dysmorphic features.JGM and DMA received predoctoral contracts from the Ministry of Education of Spain (FPI grant BES-2016-077901). This work was supported by Grant PID2019-104763RB-I00 and Ramón y Cajal contract (RYC-2014-15991), both from MINECO/AEI/FEDER (EU) to EP; and grants from the European Commission Seventh Framework Programme (ERA-NET NEURON8-Full-815-094), AEI-MICIU/FEDER (RTI2018-095582-B-I00 and RED2018-102723- T), and the iLUNG programme from the Comunidad de Madrid (B2017/BMD-3884) to MM. CNIO is a Severo Ochoa Center of Excellence (AEI-MICIU CEX2019-000891-S)

Gene expression profiling of mouse p53-deficient epidermal carcinoma defines molecular determinants of human cancer malignancy

<p>Abstract</p> <p>Background</p> <p>The epidermal specific ablation of <it>Trp53 </it>gene leads to the spontaneous development of aggressive tumors in mice through a process that is accelerated by the simultaneous ablation of <it>Rb </it>gene. Since alterations of p53-dependent pathway are common hallmarks of aggressive, poor prognostic human cancers, these mouse models can recapitulate the molecular features of some of these human malignancies.</p> <p>Results</p> <p>To evaluate this possibility, gene expression microarray analysis was performed in mouse samples. The mouse tumors display increased expression of cell cycle and chromosomal instability associated genes. Remarkably, they are also enriched in human embryonic stem cell gene signatures, a characteristic feature of human aggressive tumors. Using cross-species comparison and meta-analytical approaches, we also observed that spontaneous mouse tumors display robust similarities with gene expression profiles of human tumors bearing mutated TP53, or displaying poor prognostic outcome, from multiple body tissues. We have obtained a 20-gene signature whose genes are overexpressed in mouse tumors and can identify human tumors with poor outcome from breast cancer, astrocytoma and multiple myeloma. This signature was consistently overexpressed in additional mouse tumors using microarray analysis. Two of the genes of this signature, AURKA and UBE2C, were validated in human breast and cervical cancer as potential biomarkers of malignancy.</p> <p>Conclusions</p> <p>Our analyses demonstrate that these mouse models are promising preclinical tools aimed to search for malignancy biomarkers and to test targeted therapies of prospective use in human aggressive tumors and/or with p53 mutation or inactivation.</p

Development of anti-membrane type 1-matrix metalloproteinase nanobodies as immunoPET probes for triple negative breast cancer imaging

14 p.-6 fig.1 tab.Triple-negative breast cancer (TNBC) is characterized by aggressiveness and high rates of metastasis. The identification of relevant biomarkers is crucial to improve outcomes for TNBC patients. Membrane type 1-matrix metalloproteinase (MT1-MMP) could be a good candidate because its expression has been reported to correlate with tumor malignancy, progression and metastasis. Moreover, single-domain variable regions (VHHs or Nanobodies) derived from camelid heavy-chain-only antibodies have demonstrated improvements in tissue penetration and blood clearance, important characteristics for cancer imaging. Here, we have developed a nanobody-based PET imaging strategy for TNBC detection that targets MT1-MMP. A llama-derived library was screened against the catalytic domain of MT1-MMP and a panel of specific nanobodies were identified. After a deep characterization, two nanobodies were selected to be labeled with gallium-68 (68Ga). ImmunoPET imaging with both ([68Ga]Ga-NOTA-3TPA14 and [68Ga]Ga-NOTA-3CMP75) in a TNBC mouse model showed precise tumor-targeting capacity in vivo with high signal-to-background ratios. (68Ga)Ga-NOTA-3CMP75 exhibited higher tumor uptake compared to (68Ga)Ga-NOTA-3TPA14. Furthermore, imaging data correlated perfectly with the immunohistochemistry staining results. In conclusion, we found a promising candidate for nanobody-based PET imaging to be further investigated as a diagnostic tool in TNBC.This research was supported by BBVA Foundation grants for Scientific Research Teams: “Imaging of triple-negative breast cancer with specific miniaturized antibodies by ImmunoPET (BREIMPET)” Ref.:PR[17]_BIO_IMG_0114 (2017) and “Radioinmunotheragnostics for metastatic lung cancer with pretargeted clickable Ab Fragments (TherAbnostic)” Ref.: PR[19]_BIO_IMG_0096. (2020).Peer reviewe

Patient-derived olfactory mucosa for study of the non-neuronal contribution to amyotrophic lateral sclerosis pathology

Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease which currently has no cure. Research using rodent ALS models transgenic for mutant superoxide dismutase 1 (SOD1) has implicated that glial-neuronal interactions play a major role in the destruction of motor neurons, but the generality of this mechanism is not clear as SOD1 mutations only account for less than 2% of all ALS cases. Recently, this hypothesis was backed up by observation of similar effects using astrocytes derived from post-mortem spinal cord tissue of ALS patients which did not carry SOD1 mutations. However, such necropsy samples may not be easy to obtain and may not always yield viable cell cultures. Here, we have analysed olfactory mucosa (OM) cells, which can be easily isolated from living ALS patients. Disease-specific changes observed when ALS OM cells were co-cultured with human spinal cord neurons included decreased neuronal viability, aberrant neuronal morphology and altered glial inflammatory responses. Our results show the potential of OM cells as new cell models for ALS.This work was supported by grant SAF2010-22106 from the Spanish Ministry of Science and by the agency Pedro Laín Entralgo for neurodegenerative diseases research (2007, NDG07/6).Peer Reviewe

Efficient expression of bioactive murine IL12 as a self-processing P2A polypeptide driven by inflammation-regulated promoters in tumor cell lines

Interleukin 12 (IL12) is a heterodimeric proinflammatory cytokine that has shown promise as an anticancer agent. However, despite encouraging results in animal models, clinical trials involving IL12 have been unsuccessful due to toxic side effects associated with its systemic administration, prompting investigation into new delivery methods to confine IL12 expression to the tumor environment. In this study we used the self-cleaving property of the 2A peptide to express both codon-optimized murine IL12 subunits (muIL12opt) as a self-processing polypeptide (muIL12opt-P2A). We cloned muIL12opt-P2A driven by different inflammation-induced lentiviral expression systems to transduce murine tumor cell lines commonly employed in syngeneic tumor models. We confirmed the inducibility of these systems in vitro and in vivo and demonstrated the successful expression of both IL12 subunits and the release of bioactive IL12 upon proinflammatory stimulation in vitro. Therefore, IL12 release driven by these inflammation-regulated expression systems might be useful not only to address the impact of IL12 expression in the tumor environment but also to achieve a local IL12 release controlled by the inflammation state of the tumor, thus avoiding toxic side effects associated with systemic administration.A.R. is supported by the Spanish Ministry of Economy and Competitivity (MINECO; SAF2012-32166) and the Comunidad Autónoma de Madrid (S2010/BMD-2312). JMZ is supported by the Instituto de Salud de Carlos III (PI12/01135).Peer Reviewe

Intratumoral expression using a NFkB-based promoter enhances IL12 antitumor efficacy

Interleukin 12 is a promising anti-cancer agent; however, IL12 systemic administration is hampered by side-effects. Although intratumoral administration of IL12 is giving promising results in clinical trials, only a small percentage of patients show a complete therapeutic response. This outcome could be improved by controlling the IL12 expression window. In this work we have tested the efficacy of a self-processing P2A and codon optimized murine IL12 (mIL12Pop) using inflammation-regulated lentivectors in a syngeneic tumor model. Our results show that implantation of cells expressing mIL12Pop employing either the strong constitutive SFFV promoter or a NFkB-based promoter reduced tumor growth, caused CD8+ T cell activation and increased IFNγ production. Importantly, the use of NFkBp-mIL12Pop increased the number of CD8+ TILs and improved the remission rate without increasing IL12-serum concentration. Further experiments suggest that there is a threshold intratumoral IL12 concentration that must be reached to trigger an efficient antitumor response and a limit that once surpassed causes detrimental systemic side effects. Altogether, these results demonstrate that using NFKBp-mIL12Pop significantly increases the overall survival of the mice. In summary, this new inflammation-regulated expression system might be useful for the development of new IL12 delivery systems with improved anti-tumor activity and limited toxicity.AR is supported by the Spanish Ministry of Economy and Competitiveness (MINECO; SAF2012-32166) and the Comunidad Autonoma de Madrid, Spain (S2010/BMD-2312). FM is supported by the Comunidad Autonoma de Madrid, Spain (S2017/BMD-3867) and co-financed by European Structural and Investment Funds. AA and JMZ are supported by the Instituto de Salud Carlos III, Spain (PI15/01491 and PI16/00895, respectively). H.A. holds a research fellowship from Spanish Ministry of Education, Culture and Sports (MECD).Peer reviewe