Vector lentiviral de expresión autolimitada

La presente invención se refiere a un vector lentivírico biscistrónico de expresión autolimitada, a un sistema de producción de dicho vector lentivírico así como a los usos de dicho vector en terapia, en particular en osteogénesis.REIVINDICACIONES: 1.Una secuencia polinucleotídica que comprende: a. un polinucleótido de interés que codifica para un mediador primario intracelular y un primer promotor eucariota, en el que el primer promotor eucariota controla la expresión de dicho polinucleótido de interés, y b. un polinucleótido que codifica para la proteína CRE recombinasa y un segundo promotor eucariota, en el que el segundo promotor controla la expresión de la proteína Cre recombinasa y en el que el segundo promotor eucariota queda activado por un producto de expresión del polinucleótido de interés del paso a) con lo que se produce un control endógeno de la expresión de dicho polinucleótido, donde por mediador primario intracelular se entiende el codificado de forma directa por el polinucleótido de interés, donde por producto de expresión se entiende un mediador secundario intracelular, donde por mediador secundario intracelular se entiende un mediador sintetizado por una célula diana como consecuencia del proceso puesto en marcha por la expresión del mediador primario y donde por segundo promotor se entiende un promotor específico de respuesta al mediador secundario intracelular. 2.La secuencia polinucleotídica de la reivindicación 1, donde dicha secuencia es un casete de expresión. 3. Un vector que comprende la secuencia polinucleotídica de la reivindicación 1. 4. Un vector viral que comprende la secuencia polinucleotídica de la reivindicación 1 o el casete de expresión de la reivindicación 2. 5. El vector viral de la reivindicación 4, donde dicho vector es un vector lentiviral bicistrónico recombinante de expresión autolimitada capaz de transducir una célula diana, donde por vector de expresión autolimitada se entiende aquél que contiene elementos que permiten eliminar el provirus del genoma de la célula diana de forma regulada endógenamente mediante la acción de un mediador secundario sintetizado por la célula diana en respuesta a la expresión del polinucleótido de interés. 6. Un vector lentivírico bicistrónico de expresión autolimitada recombinante que comprende: a. El conjunto de secuencias nucleotídicas que codifican las proteínas y demás factores requeridos implicados en la regulación postranscripcional, así como en el empaquetamiento del virus, donde dichas secuencias se seleccionan de la lista que consiste en: RRE (Rev Response Element) ; cPPT (central Polypurine Track) y WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulator y Element) , b. Un cistrón que comprende un polinucleótido de interés que codifica para un mediador primario intracelular y un primer promotor eucariota, en el que el primer promotor eucariota controla la expresión de dicho polinucleótido de interés, y c. un segundo cistrón que comprende un polinucleótido que codifica para la proteína Cre recombinasa y un segundo promotor eucariota, en el que el segundo promotor controla la expresión de la proteína Cre recombinasa y en el que el segundo promotor eucariota queda activado por un producto de expresión del polinucleótido de interés del paso a) con lo que se produce un control endógeno de la expresión de dicho polinucleótido, y d. donde dicho vector comprende la presencia de dos regiones loxP flanqueando el provirus tras la integración del DNA en la célula diana, donde por mediador primario intracelular se entiende el codificado de forma directa por el polinucleótido de interés, donde por producto de expresión se entiende un mediador secundario intracelular, preferiblemente implicado en la diferenciación celular, donde por mediador secundario intracelular implicado en la diferenciación celular se entiende un mediador sintetizado por una célula diana como consecuencia del proceso puesto en marcha por la expresión del mediador primario y donde por segundo promotor se entiende un promotor específico de respuesta al mediador secundario intracelular. 7.El polinucleótido de la reivindicación 1, el casete de expresión de la reivindicación 2 o el vector de cualquiera de las reivindicaciones 3 a 4, en el que el gen de interés codifica para un mediador primario intracelular implicado en la diferenciación y el producto de expresión es un mediador secundario intracelular implicado en la diferenciación. 8. El polinucleótido de la reivindicación 1, el casete de expresión de la reivindicación 2 o el vector de cualquiera de las reivindicaciones 3 a 6, en el que el gen de interés codifica para un mediador primario intracelular implicado en la diferenciación osteogénica y el producto de expresión es un mediador secundario intracelular implicado en la diferenciación osteogénica. 9. El polinucleótido de la reivindicación 1, el casete de expresión de la reivindicación 2 o el vector de cualquiera de las reivindicaciones 3 a 6, en el que el gen de interés es el Dlx5 (Sec. Nucleotídica: NM_005221; Sec. Proteica: NP_005212.1) y donde el producto de expresión es Osterix (Sec. Nucleotídica: AF477981; Sec. Proteica: AAL84281.1) 10. El polinucleótido de la reivindicación 1, el casete de expresión de la reivindicación 2 o el vector de cualquiera de las reivindicaciones 3 a 6, en el que el gen de interés heterólogo es el Dlx5, el producto de expresión es Osterix (Sec. Nucleotídica: AF477981; Sec. Proteica: AAL84281.1) y donde el segundo promotor es el promotor de Satb2 y el primer promotor es el promotor EF-1. 11. El polinucleótido, el casete de expresión o el vector según cualquiera de las reivindicaciones precedentes, que además comprende un elemento regulador posttranscripcional o un elemento traduccional. 12. Un sistema de producción de vector vírico para producir una partícula de vector derivada de un virus comprendiendo dicho sistema una secuencia de ácido nucleico o un conjunto de secuencias de ácido nucleico que codifican los componentes del vector incluyendo las secuencias de ácido nucleico tal y como se han definido éstas en una cualquiera de las reivindicaciones 1-2 y 7-11. 13. El sistema de producción según la reivindicación 12, donde dicho sistema de producción es el sistema de producción de un vector lentivírico y en el que la secuencia de ácido nucleico o el conjunto de secuencias de ácido nucleico comprende tres constructos de ADN que codifican (i) los componentes del vector incluyendo las secuencias de ácido nucleico tal y como se han definido éstas en cualquiera de la reivindicaciones precedentes, (ii) Gag y Pol y Rev y (iii) Env o (ii) Gag y Pol, (iii) Rev y (iv) Env. 14. Un proceso para preparar una partícula de vector viral que comprende introducir la secuencia de ácido nucleico o el conjunto de secuencias de ácido nucleico tal y como se definen en una cualquiera de las reivindicaciones 1-2 y 7-11, en una célula hospedadora, y obtener una partícula de vector viral. 15. Una partícula de vector viral o lentivírico producida mediante el sistema de cualquiera de las reivindicaciones 12 o 13 o producida mediante el proceso según la reivindicación 14. 16. Una célula transducida o transformada con el vector de cualquiera de las reivindicaciones 3-6 o con la partícula de vector viral o lentivírico de la reivindicación 15. 17. La célula transducida o transformada de la reivindicación 16 o el vector de cualquiera de las reivindicaciones 3-6 o la partícula de vector viral o lentivírico de la reivindicación 15, para uso en terapia. 18. Una composición farmacéutica que comprende la célula transducida o transformada de la reivindicación 16 o el vector de cualquiera de las reivindicaciones 3-6 o la partícula de vector viral o lentivírico de la reivindicación 15. 19. La composición farmacéutica de la reivindicación 18 o la célula transducida o transformada de la reivindicación 16 o el vector de cualquiera de las reivindicaciones 3-6 o la partícula de vector viral o lentivírico de la reivindicación 15, para suministrar un gen de interés a un sitio diana necesitado del mismo. 20. La composición farmacéutica de la reivindicación 18 o la célula transducida o transformada de la reivindicación 16 o el vector de cualquiera de las reivindicaciones 3-6 o la partícula de vector viral o lentivírico de la reivindicación 15, para su uso en osteogénesis o en la regeneración ósea.Cuando una patente se hace internacional, se puede encontrar en el idioma de cada país en que se ha solicitado. En Espacenet se tiene acceso a los documentos en cada idioma.Instituto de Salud Carlos IIISolicitud de patent

Extracellular Vesicle-Mediated Immune Regulation of Tissue Remodeling and Angiogenesis After Myocardial Infarction

Myocardial ischemia-related disorders constitute a major health problem, being a leading cause of death in the world. Upon ischemia, tissue remodeling processes come into play, comprising a series of inter-dependent stages, including inflammation, cell proliferation and repair. Neovessel formation during late phases of remodeling provides oxygen supply, together with cellular and soluble components necessary for an efficient myocardial reconstruction. Immune system plays a central role in processes aimed at repairing ischemic myocardium, mainly in inflammatory and angiogenesis phases. In addition to cellular components and soluble mediators as chemokines and cytokines, the immune system acts in a paracrine fashion through small extracellular vesicles (EVs) release. These vesicular structures participate in multiple biological processes, and transmit information through bioactive cargoes from one cell to another. Cell therapy has been employed in an attempt to improve the outcome of these patients, through the promotion of tissue regeneration and angiogenesis. However, clinical trials have shown variable results, which put into question the actual applicability of cell-based therapies. Paracrine factors secreted by engrafted cells partially mediate tissue repair, and this knowledge has led to the hypothesis that small EVs may become a useful tool for cell-free myocardial infarction therapy. Current small EVs engineering strategies allow delivery of specific content to selected cell types, thus revealing the singular properties of these vesicles for myocardial ischemia treatment.This work was supported by grants to AA-S (FIS PI15/01491) and to FS-M (grants SAF2014-55579-R and SAF2017-82886-R to FS-M), BIOIMID PIE13/041 and CIBER CARDIOVASCULAR from the Instituto de Salud Carlos III (Fondo de Investigación Sanitaria del Instituto de Salud Carlos III with co-funding from the Fondo Europeo de Desarrollo Regional; FEDER), Programa de Actividades en Biomedicina de la Comunidad de Madrid-B2017/BMD-3671-INFLAMUNE to FS-M, and ERC2011-AdG294340-GENTRIS to FS-M, and Fundació La Marató TV3 (20152330 31). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the ProCNIC Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505).S

Extracellular Vesicle-Mediated Immune Regulation of Tissue Remodeling and Angiogenesis After Myocardial Infarction

Myocardial ischemia-related disorders constitute a major health problem, being a leading cause of death in the world. Upon ischemia, tissue remodeling processes come into play, comprising a series of inter-dependent stages, including inflammation, cell proliferation and repair. Neovessel formation during late phases of remodeling provides oxygen supply, together with cellular and soluble components necessary for an efficient myocardial reconstruction. Immune system plays a central role in processes aimed at repairing ischemic myocardium, mainly in inflammatory and angiogenesis phases. In addition to cellular components and soluble mediators as chemokines and cytokines, the immune system acts in a paracrine fashion through small extracellular vesicles (EVs) release. These vesicular structures participate in multiple biological processes, and transmit information through bioactive cargoes from one cell to another. Cell therapy has been employed in an attempt to improve the outcome of these patients, through the promotion of tissue regeneration and angiogenesis. However, clinical trials have shown variable results, which put into question the actual applicability of cell-based therapies. Paracrine factors secreted by engrafted cells partially mediate tissue repair, and this knowledge has led to the hypothesis that small EVs may become a useful tool for cell-free myocardial infarction therapy. Current small EVs engineering strategies allow delivery of specific content to selected cell types, thus revealing the singular properties of these vesicles for myocardial ischemia treatment

New targeting agent for the selective drug delivery of nanocarriers for treating neuroblastoma

Novel targeting agents against neuroblastoma based on the meta-iodobenzylguanidine (MIBG) moiety were synthesized and biologically evaluated for nanocarrier vectorization. These compounds have been anchored on the surface of drug loaded mesoporous silica nanocarriers, resulting in the improved cellular uptake in tumoral cells. Neuroblastoma (NB) is the most frequent extracranial pediatric tumor. Advanced forms of the disease (metastatic and/or refractory) have a dismal prognosis despite the combination of chemotherapy, radiotherapy, surgery and bone narrow transplants. These treatments carry severe side effects and, in some cases, compromise the life of the patient. MIBG has been widely applied in the medical diagnosis of NB due to its affinity for tumor cells through the norepinephrine transporter (NET), which is expressed in 90% of NB tumors. The exclusive accumulation of MIBG in neuroblastoma has been widely studied; however, its properties have been never exploited as a targeting agent in nanocarrier drug delivery systems. Several structural analogues of MIBG have been prepared and attached on the surface of nanocarriers. Their selective internalization has been tested against human neuroblastoma cells, which show, in the best case, cellular uptake four times higher than that of the naked nanosystem. Furthermore, in vivo experiments showed preferential and selective accumulation and retention of the targeted nanosystem comparing with the naked and only PEGylated counterpart systems. This novel nanosystem could be easily applicable to all kinds of drug delivery nanocarriers, providing a universal tool for neuroblastoma chemotherapies that is superior to classical approaches through a novel nanosystem exclusively designed to target this terrible malignancy

When should we order a next generation sequencing test in a patient with cancer?

Technical advances in genome sequencing and the implementation of next-generation sequencing (NGS) in clinical oncology have paved the way for individualizing cancer patient therapy based on molecular profiles. When and how to use NGS testing in the clinic is at present an unsolved issue, although new research results provide evidence favoring this approach in some types of advanced cancer. Clinical research is evolving rapidly, from basket and umbrella trials to adaptative design precision oncology clinical studies, and genomic and molecular data often displace the classical clinical validation procedures of biomarkers. In this context, physicians must be aware of the clinical evidence behind these new biomarkers and NGS tests available, in order to use them in the right moment, and with a critical point of view. This review will present the status of currently available targeted drugs that can be effective based on actionable molecular alterations, and the NGS tests that are currently available, offering a practical guide for the application of Clinical Precision Oncology in the real world routine practice.probability of identifying a targetable mutation is low[62], the canceris in early stages with recognized and effective forms of standardtreatment, or the patient has an irreversible disease with very shortlife-expectancy. As with any other laboratory test, doctors andpatients must be sure before ordering an NGS test that its result willhave an impact of the therapeutic plan. In any case, standard single-gene molecular testing must always be performed when indicated,since important therapeutic targets might be potentially missed if nomolecular analyses were performed.Clinical trials are showing that NGS testing can have an impact inthe response rate and progression-free survival of patients, and cantherefore be a very useful strategy leading to new molecularly-tar-geted treatment indications. Key factors responsible for improvedresults in precision-oriented clinical research, include refining themolecular pathways studied, developing molecular testing that inte-grates standarised genomic tests with transcriptomic analysis andimmunohistochemistry, selecting more active targeted agents,designing combinations of targeted agents -also with other forms oftherapy, and providing early treatment recommendations with avail-able Molecular Multidisciplinary Boards. Interdisciplinary discussionare very important to help with the interpretation of unclear molecu-lar results that are oftentimes seen with NGS testing.Important unsolved issues that will need to be addressed in thefuture include deciding which is the best tissue to perform NGS (pri-mary tumor vs metastasis, tumor DNA vs circulating tumor DNA),when is the right moment to test (atfirst diagnosis of advanced dis-ease or when the disease is refractory), and whether there are NGSclinical trial designs that allow for the use of control groups. Finally,using a complete informed consent before NGS testing and communi-cating NGS reports to patients are two very important aspects of theprocedure that have raised ethical concerns, and that must be alwaysaddressed by the practicing oncologists when ordering a NGS test.Search strategy and selection criteriaWe identified references through PubMed with the search terms“cancer AND NGS,”“cancer AND next generation sequencing,”“can-cer AND genomics,”for articles published to March 30, 2020. Thefinalreference list was generated on the basis of originality and relevanceto the broad scope of this Review.FundingThis Review was funded in part by research funds from projectsPIE15/00068andPI17/01865(Instituto de Salud Carlos III) awardedto RC, projectsJR17/00007andPI17/008(Instituto de Salud CarlosIII), awarded to NR-L,PI15/01491andPI19/00549(Instituto de SaludCarlos III) awarded to AA, projectsSAF2017 82886-R(Ministerio deEconomía y Competitividad), INDISNET-S2011/BMD-2332(Fundaci on Ram on Areces), andHR17-00016("La Caixa" Foundation)awarded to FS-M, and projectsPI16/00354(Instituto de Salud CarlosIII) andB2017/BMD-3733from the Consejería de Educaci on, Juventudy Deporte, Comunidad de Madrid, awarded to MQ-F. The manuscriptis part of the activities of the endowed Chair of Personalised PrecisionOncology, Universidad Aut onoma de Madrid (UAM-Fundaci on Insti-tuto Roche)S

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

Clonal dynamics in osteosarcoma defined by RGB marking

Osteosarcoma is a type of bone tumour characterized by considerable levels of phenotypic heterogeneity, aneuploidy, and a high mutational rate. The life expectancy of osteosarcoma patients has not changed during the last three decades and thus much remains to be learned about the disease biology. Here, we employ a RGB-based single-cell tracking system to study the clonal dynamics occurring in a de novo-induced murine osteosarcoma model. We show that osteosarcoma cells present initial polyclonal dynamics, followed by clonal dominance associated with adaptation to the microenvironment. Interestingly, the dominant clones are composed of subclones with a similar tumour generation potential when they are re-implanted in mice. Moreover, individual spontaneous metastases are clonal or oligoclonal, but they have a different cellular origin than the dominant clones present in primary tumours. In summary, we present evidence that osteosarcomagenesis can follow a neutral evolution model, in which different cancer clones coexist and propagate simultaneously.We thank ISCIII and CNIO flow cytometry and cell sorting units for their participation in our studies. We are thankful to the CCEH-Fred Hutchinson Cancer Research Center for LAM-PCR service. We acknowledge Raquel Pérez Tavarez, María Blázquez Mesa, Alicia Giménez Sánchez, Elena Calvo Cazalilla, and Monserrat Arroyo Correas for useful help on the pathology studies; and Teresa Cejalvo, Isabel Cubillo Moreno, and Miguel Angel Rodríguez-Milla for their contributions in experimental setup. We thank the visual artist Isabella Lacquaniti for her help with drawings and schematics. We are also thankful to the Fondo de Investigaciones Sanitarias (FIS: PI11/00377 and PI14CIII/00005 to J.G.-C., FIS: CP11/00206 to A.A., and RTICC: RD12/0036/0027 to J.G.-C.), the Madrid Regional Government (CellCAM; P2010/BMD-2420 to J.G.-C.), the Asociación Pablo Ugarte, and the Asociación Afanion for grants support.S

A new role for circulating T follicular helper cells in humoral response to anti-PD-1 therapy

Background Lung cancer is one of the most frequent malignancies in humans and is a major cause of death. A number of therapies aimed at reinforcing antitumor immune response, including antiprogrammed cell death protein 1 (anti-PD-1) antibodies, are successfully used to treat several neoplasias as non-small cell lung cancer (NSCLC). However, host immune mechanisms that participate in response to anti-PD-1 therapy are not completely understood. Methods We used a syngeneic immunocompetent mouse model of NSCLC to analyze host immune response to anti-PD-1 treatment in secondary lymphoid organs, peripheral blood and tumors, by flow cytometry, immunohistochemistry and quantitative real-time PCR (qRT-PCR). In addition, we also studied specific characteristics of selected immune subpopulations in ex vivo functional assays. Results We show that anti-PD-1 therapy induces a population of circulating T follicular helper cells (cTfh) with enhanced B activation capacity, which participates in tumor response to treatment. Anti-PD-1 increases the number of tertiary lymphoid structures (TLS), which correlates with impaired tumor growth. Of note, TLS support cTfh-associated local antibody production, which participates in host immune response against tumor. Conclusion These findings unveil a novel mechanism of action for anti-PD-1 therapy and provide new targets for optimization of current therapies against lung cancer.This work was supported by grants to AA: FIS PI15/01491 and CIBER CARDIOVASCULAR from the Instituto de Salud Carlos III (Fondo de Investigación Sanitaria del Instituto de Salud Carlos III with co-funding from the Fondo Europeo de Desarrollo Regional; FEDER

c-Fos induces chondrogenic tumor formation in immortalized human mesenchymal progenitor cells

Mesenchymal progenitor cells (MPCs) have been hypothesized as cells of origin for sarcomas, and c-Fos transcription factor has been showed to act as an oncogene in bone tumors. In this study, we show c-Fos is present in most sarcomas with chondral phenotype, while multiple other genes are related to c-Fos expression pattern. To further define the role of c-Fos in sarcomagenesis, we expressed it in primary human MPCs (hMPCs), immortalized hMPCs and transformed murine MPCs (mMPCs). In immortalized hMPCs, c-Fos expression generated morphological changes, reduced mobility capacity and impaired adipogenic- and osteogenic-differentiation potentials. Remarkably, immortalized hMPCs or mMPCs expressing c-Fos generated tumors harboring a chondrogenic phenotype and morphology. Thus, here we show that c-Fos protein has a key role in sarcomas and that c-Fos expression in immortalized MPCs yields cell transformation and chondrogenic tumor formation.This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS: PI11/00377 to J.G.-C.; and RTICC: RD12/0036/0027 to J.G-C, RD12/0036/0020 to S.M.) and the Madrid Regional Government (CellCAM; P2010/BMD-2420 to J.G.-C) in Spain. A.A. was supported by Juan de la Cierva program of the Spanish Plan Nacional (MINECO) and Sara Borrell program of the ISCIII/FEDER. A.Al. was supported by the “Miguel Servet” program of the ISCIII/FEDER. We gratefully acknowledge support from Asociación Pablo Ugarte (CIF G86121019) and AFANION (CIF G02223733). The experiments were approved by the appropriate committees.S

Peripheral Blood Mononuclear Cells Predict Therapeutic Efficacy of Immunotherapy in NSCLC

In lung cancer immunotherapy, biomarkers to guide clinical decisions are limited. We now explore whether the detailed immunophenotyping of circulating peripheral blood mononu-clear cells (PBMCs) can predict the efficacy of anti-PD-1 immunotherapy in patients with advanced non-small-cell lung cancer (NSCLC). We determined 107 PBMCs subpopulations in a prospective cohort of NSCLC patients before starting single-agent anti-PD-1 immunotherapy (study group), an-alyzed by flow cytometry. As a control group, we studied patients with advanced malignancies before initiating non-immunotherapy treatment. The frequency of PBMCs was correlated with treatment outcome. Patients were categorized as having either high or low expression for each bi-omarker, defined as those above the 55th or below the 45th percentile of the overall marker expres-ion within the cohort. In the study group, three subpopulations were associated with significant differences in outcome: high pretreatment levels of circulating CD4+CCR9+, CD4+CCR10+, or CD8+CXCR4+ T cells correlated with poorer overall survival (15.7 vs. 35.9 months, HR 0.16, p = 0.003; 22.0 vs. NR months, HR 0.10, p = 0.003, and 22.0 vs. NR months, HR 0.29, p = 0.02). These differences were specific to immunotherapy-treated patients. High baseline levels of circulating T cell subpopulations related to tissue lymphocyte recruitment are associated with poorer outcomes of immunotherapy-treated advanced NSCLC patientsProjects PIE15/00068, PI17/01865, and PI20/01458 (Instituto de Salud Carlos III) awarded to R.C.; Projects FIS PI19/01491 and CIBER Cardiovascular (Fondo de Investigación Sanitaria del Instituto de Salud Carlos III with co-funding from the Fondo Europeo de Desarrollo Regional FEDER) awarded to A.A.; CNIO Bioinformatics Unit is supported by the Instituto de Salud Carlos III (ISCIII); Project RETOS RTI2018-097596-B-I00 (AEI/10.13039/501100011033 MCI/FEDER, UE); Projects PI17/00801 and PI21/01111 grants from Instituto de Salud Carlos III and JR17/00007 awarded to N.R.-L., and Project Molecular Analysis of the Exhaled Breath Condensate in the Management of Solitary Pulmonary Nodule (ideas semilla AECC 2019), from Asociación Española Contra el Cáncer (AECC), awarded to J.