Rodriguez-Madoz, J.R. (Juan Roberto)
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- Generation of an induced pluripotent stem cell line (CIMAi001-A) from a compound heterozygous Primary Hyperoxaluria Type I (PH1) patient carrying p.G170R and p.R122* mutations in the AGXT gene.(Elsevier BV, 2019) Rodriguez-Marquez, P. (Paula); Rodriguez-Madoz, J.R. (Juan Roberto); Rodriguez, S. (Saray); Martin-Mallo, A. (Angel); Martínez-Turrillas, R. (Rebeca); Beck, B.B. (Bodo B.); Salido, E. (Eduardo); Prosper-Cardoso, F. (Felipe)Primary Hyperoxaluria Type I (PH1) is a rare autosomal recessive metabolic disorder characterized by defects in enzymes involved in glyoxylate metabolism. PH1 is a life-threatening disease caused by the absence, deficiency or mistargeting of the hepatic alanine-glyoxylate aminotransferase (AGT) enzyme. A human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a PH1 patient being compound heterozygous for the most common mutation c.508G>A (G170R), a mistargeting mutation, and c.364C>T (R122*), a previously reported nonsense mutation in AGTX. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development.
- Preclinical evaluation of a cell-based gene therapy using the sleeping beauty transposon system in choroidal neovascularization(Elsevier BV, 2019) Ivics, Z. (Zoltán); Garcia-Garcia, L. (Laura); Marie, C. (Corinne); Johnen, S. (S.); Rodriguez-Madoz, J.R. (Juan Roberto); Scherman, D. (Daniel); Pouillot, S. (Severine); Garcia-Layana, A. (Alfredo); Izsvák, Z. (Zsuzsanna); Thumann, G. (Gabriele); Diarra, S. (Sabine); Bezunartea, J. (Jaione); Miskey, C. (Csaba); Fernandez-Robredo, P. (Patricia); Sebe, A. (Attila); Prosper-Cardoso, F. (Felipe); Kropp, M. (Martina); Hernandez, M. (María); Recalde, S. (Sergio)Age-related macular degeneration (AMD) is a progressive retinal disorder characterized by imbalanced pro- and antiangiogenic signals. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells were co-transfected with pFAR4-inverted terminal repeats (ITRs) CMV-PEDF-BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats, and transfected primary cells (transfected RPE [tRPE] and transfected IPE [tIPE] cells) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinases 2 and 9 (MMP-2/9), and microglial/macrophage markers were measured. Injection with tRPE/IPE cells significantly reduced the leakage area at 7 and 14 days and the CNV area at 7 days. There was a significant increase in PEDF and the PEDF/VEGF ratio with tRPE cells and a reduction in the MMP-2 activity. Our data demonstrated that ex vivo non-viral gene therapy reduces CNV and could be an effective and safe therapeutic option for angiogenic retinal diseases.
- Revealing Cell Populations Catching The Early Stages Of Human Embryo Development In Naive Pluripotent Stem Cell Cultures(2023) Ullate-Agote, A. (Asier); Rodriguez-Madoz, J.R. (Juan Roberto); Garate-Iturriagagoitia, L. (Leire); Aranguren-López, X. (Xabier); Barreda, C. (Carolina); Coppiello, G. (Giulia); Romero-Riojas, J.P. (Juan Pablo); Carvajal-Vergara, X. (Xonia); Aguirre-Ena, X. (Xabier); Dupéré-Richer, D. (Daphné); Prosper, F. (Felipe); Moya-Jódar, M. (Marta); Barlabé-Ginesta, P. (Paula); Abizanda-Sarasa, G. (Gloria)Naive human pluripotent stem cells (hPSCs) are defined as the in vitro counterpart of the human preimplantation embryo's epiblast and are used as a model system to study developmental processes. In this study, we report the discovery and characterization of distinct cell populations coexisting with epiblast-like cells in 5iLAF naive human induced PSC (hiPSC) cultures. It is noteworthy that these populations closely resemble different cell types of the human embryo at early developmental stages. While epiblast-like cells represent the main cell population, interestingly we detect a cell population with gene and transposable element expression profile closely resembling the totipotent eight-cell (8C)-stage human embryo, and three cell populations analogous to trophectoderm cells at different stages of their maturation process: transition, early, and mature stages. Moreover, we reveal the presence of cells resembling primitive endoderm. Thus, 5iLAF naive hiPSC cultures provide an excellent opportunity to model the earliest events of human embryogenesis, from the 8C stage to the peri-implantation period.
- Targeting the extra domain A of fibronectin for cancer therapy with CAR-T cells(2022) Vilas, A. (Amaia); Sarrión, P. (Patricia); Conde-Gallastegi, E. (Enrique); Sánchez-Moreno, I. (Inés); Rodriguez-Madoz, J.R. (Juan Roberto); Lozano-Moreda, T. (Teresa); Hervas-Stubbs, S. (Sandra); Casares, N. (Noelia); Navarro-Negredo, F.C. (Flor Cecilia); Echeveste, J.I. (José I.); Martín-Otal, C. (Celia); Gorraiz, M. (Marta); Andrea, C.E. (Carlos Eduardo) de; Serrano-Tejero, D. (Diego); Lasarte-Cia, A. (Aritz); Prosper-Cardoso, F. (Felipe); Calvo-González, A. (Alfonso); San-Miguel, J.F. (Jesús F.); Lasarte, J.J. (Juan José)Background One of the main difficulties of adoptive cell therapies with chimeric antigen receptor (CAR)-T cells in solid tumors is the identification of specific target antigens. The tumor microenvironment can present suitable antigens for CAR design, even though they are not expressed by the tumor cells. We have generated a CAR specific for the splice variant extra domain A (EDA) of fibronectin, which is highly expressed in the tumor stroma of many types of tumors but not in healthy tissues. Methods EDA expression was explored in RNA-seq data from different human tumor types and by immunohistochemistry in paraffin-embedded tumor biopsies. Murine and human anti-EDA CAR-T cells were prepared using recombinant retro/lentiviruses, respectively. The functionality of EDA CAR-T cells was measured in vitro in response to antigen stimulation. The antitumor activity of EDA CAR-T cells was measured in vivo in C57BL/6 mice challenged with PM299L-EDA hepatocarcinoma cell line, in 129Sv mice-bearing F9 teratocarcinoma and in NSG mice injected with the human hepatocarcinoma cell line PLC. Results EDA CAR-T cells recognized and killed EDA-expressing tumor cell lines in vitro and rejected EDA-expressing tumors in immunocompetent mice. Notably, EDA CAR-T cells showed an antitumor effect in mice injected with EDA-negative tumor cells lines when the tumor stroma or the basement membrane of tumor endothelial cells express EDA. Thus, EDA CAR-T administration delayed tumor growth in immunocompetent 129Sv mice challenged with teratocarcinoma cell line F9. EDA CAR-T treatment exerted an antiangiogenic effect and significantly reduced gene signatures associated with epithelial-mesenchymal transition, collagen synthesis, extracellular matrix organization as well as IL-6-STAT5 and KRAS pathways. Importantly, the human version of EDA CAR, that includes the human 41BB and CD3 zeta endodomains, exerted strong antitumor activity in NSG mice challenged with the human hepatocarcinoma cell line PLC, which expresses EDA in the tumor stroma and the endothelial vasculature. EDA CAR-T cells exhibited a tropism for EDA-expressing tumor tissue and no toxicity was observed in tumor bearing or in healthy mice. Conclusions These results suggest that targeting the tumor-specific fibronectin splice variant EDA with CAR-T cells is feasible and offers a therapeutic option that is applicable to different types of cancer.
- Chromatin activation as a unifying principle underlying pathogenic mechanisms in multiple myeloma(2020) García-Torre, B. (Beatriz); Soler-Vila, P. (Paula); Taha, R.Y. (Ruba Y.); Beekman, R. (Renée); Rodriguez-Madoz, J.R. (Juan Roberto); El-Omri, H. (Halima); Charalampopoulou, S. (Stella); San-Jose-Eneriz, E. (Edurne); Martens, J.H.A. (Joost H. A.); Flicek, P. (Paul); Agirre, X. (Xabier); Stunnenberg, H.G. (Hendrik G.); Garate, L. (Leire); Kulis, M. (Marta); Mitsiades, C.S. (Constantine S.); Licht, J.D. (Jonathan D.); Carrasco-León, A. (Arantxa); Chapaprieta, V. (Vicente); Lara-Astiaso, D. (David); Ezponda, T. (Teresa); Ordóñez-Ciriza, R. (Raquel); Verdaguer-Dot, N. (Núria); Vilas-Zornoza, A. (Amaia); Campo, E. (Elías); Dupéré-Richer, D. (Daphné); Miranda, E. (Estibaliz); Duran-Ferrer, M. (Martí); Meydan, C. (Cem); Paiva, B. (Bruno); Vilarrasa-Blasi, R. (Roser); Gut, I. (Ivo); Melnick, A. (Ari); Prosper-Cardoso, F. (Felipe); Calasanz-Abinzano, M.J. (Maria Jose); Martínez-Turrilas, R. (Rebeca); Clot, G. (Guillem); San-Miguel, J.F. (Jesús F.); Martin-Subero, J.I. (Jose Ignacio); Russiñol, N. (Nuria)Multiple myeloma (MM) is a plasma cell neoplasm associated with a broad variety of genetic lesions. In spite of this genetic heterogeneity, MMs share a characteristic malignant phenotype whose underlying molecular basis remains poorly characterized. In the present study, we examined plasma cells from MM using a multi-epigenomics approach and demonstrated that, when compared to normal B cells, malignant plasma cells showed an extensive activation of regulatory elements, in part affecting coregulated adjacent genes. Among target genes up-regulated by this process, we found members of the NOTCH, NF-kB, MTOR signaling, and TP53 signaling pathways. Other activated genes included sets involved in osteoblast differentiation and response to oxidative stress, all of which have been shown to be associated with the MM phenotype and clinical behavior. We functionally characterized MM-specific active distant enhancers controlling the expression of thioredoxin (TXN), a major regulator of cellular redox status and, in addition, identified PRDM5 as a novel essential gene for MM. Collectively, our data indicate that aberrant chromatin activation is a unifying feature underlying the malignant plasma cell phenotype.
- CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I(2018) Vilas, A. (Amaia); González-Aseguinolaza, G. (Gloria); Zabaleta-Lasarte, N. (Nerea); Betancor, I. (Isabel); Rodriguez-Madoz, J.R. (Juan Roberto); Vales, A. (África); Rodriguez, S. (Saray); Lara-Astiaso, D. (David); Martínez-Turrillas, R. (Rebeca); Castro-Labrador, L. (Laura); Olagüe, M. (María); Torella, L. (Laura); Salido, E. (Eduardo); Barberia, M. (Miren); Prosper-Cardoso, F. (Felipe); Martin-Higueras, C. (Cristina); Zapata-Linares, N.M. (Natalia María)CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1(-/-) mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites.
- Semliki forest virus expressing interleukin-12 induces antiviral and antitumoral responses in woodchucks with chronic viral hepatitis and hepatocellular carcinoma(American Society for Microbiology, 2009) Tennant, B.C. (Bud C.); González-Aseguinolaza, G. (Gloria); Rodriguez-Madoz, J.R. (Juan Roberto); Smerdou, C. (Cristian); Quetglas, J.I. (José Ignacio); Dykes, N.L. (Nathan L.); Otano, I. (Itziar); Menne, S. (Stephan); Crettaz, J. (Julien); Prieto, J. (Jesús); Butler, S.D. (Scott D.); Bellezza, C.A. (Christine A.); Ruiz-Guillen, M. (Marta); Liu, K.H. (Katherine H.)A vector based on Semliki Forest virus (SFV) expressing high levels of interleukin-12 (SFV-enhIL-12) has previously demonstrated potent antitumoral efficacy in small rodents with hepatocellular carcinoma (HCC) induced by transplantation of tumor cells. In the present study, the infectivity and antitumoral/antiviral effects of SFV vectors were evaluated in the clinically more relevant woodchuck model, in which primary HCC is induced by chronic infection with woodchuck hepatitis virus (WHV). Intratumoral injection of SFV vectors expressing luciferase or IL-12 resulted in high reporter gene activity within tumors and cytokine secretion into serum, respectively, demonstrating that SFV vectors infect woodchuck tumor cells. For evaluating antitumoral efficacy, woodchuck tumors were injected with increasing doses of SFV-enhIL-12, and tumor size was measured by ultrasonography following treatment. In five (83%) of six woodchucks, a dose-dependent, partial tumor remission was observed, with reductions in tumor volume of up to 80%, but tumor growth was restored thereafter. Intratumoral treatment further produced transient changes in WHV viremia and antigenemia, with >or=1.5-log(10) reductions in serum WHV DNA in half of the woodchucks. Antitumoral and antiviral effects were associated with T-cell responses to tumor and WHV antigens and with expression of CD4 and CD8 markers, gamma interferon, and tumor necrosis factor alpha in peripheral blood mononuclear cells, suggesting that immune responses against WHV and HCC had been induced. These experimental observations suggest that intratumoral administration of SFV-enhIL-12 may represent a strategy for treatment of chronic HBV infection and associated HCC in humans but indicate that this approach could benefit from further improvements.
- Increased efficacy and safety in the treatment of experimental liver cancer with a novel adenovirus-alphavirus hybrid vector(American Association for Cancer Research, 2006) Gomar, C. (Celia); Rodriguez-Madoz, J.R. (Juan Roberto); Smerdou, C. (Cristian); Guan, M. (Min); Qian, C. (Cheng); Kochanek, S. (Stefan); Alzuguren, P. (Pilar); Kramer, M.G. (María Gabriela); Prieto, J. (Jesús)An improved viral vector for cancer gene therapy should be capable of infecting tumors with high efficiency, inducing specific and high-level expression of transgene in the tumor and selectively destroying tumor cells. In the design of such a vector to treat hepatocellular carcinoma, we took advantage of (a) the high infectivity of adenoviruses for hepatic cells, (b) the high level of protein expression and proapoptotic properties that characterize Semliki Forest virus (SFV) replicon, and (c) tumor selectivity provided by alpha-fetoprotein (AFP) promoter. We constructed a hybrid viral vector composed of a helper-dependent adenovirus containing an SFV replicon under the transcriptional control of AFP promoter and a transgene driven by SFV subgenomic promoter. Hybrid vectors containing murine interleukin-12 (mIL-12) genes or reporter gene LacZ showed very specific and high-level expression of transgenes in AFP-expressing hepatocellular carcinoma cells, both in vitro and in an in vivo hepatocellular carcinoma animal model. Infected hepatocellular carcinoma cells were selectively eliminated due to the induction of apoptosis by SFV replication. In a rat orthotopic liver tumor model, treatment of established tumors with a hybrid vector carrying mIL-12 gene resulted in strong antitumoral activity without accompanying toxicity. This new type of hybrid vectors may provide a potent and safe tool for cancer gene therapy.
- Semliki forest virus vectors engineered to express higher IL-12 levels induce efficient elimination of murine colon adenocarcinomas(Nature, 2005) Rodriguez-Madoz, J.R. (Juan Roberto); Smerdou, C. (Cristian); Prieto, J. (Jesús)To evaluate the use of alphavirus vectors for tumor treatment we have constructed and compared two Semliki Forest virus (SFV) vectors expressing different levels of IL-12. SFV-IL-12 expresses both IL-12 subunits from a single subgenomic promoter, while in SFV-enhIL-12 each IL-12 subunit is expressed from an independent subgenomic promoter fused to the SFV capsid translation enhancer. This latter strategy provided an eightfold increase of IL-12 expression. We chose the poorly immunogenic MC38 colon adenocarcinoma model to evaluate the therapeutic potential of SFV vectors. A single intratumoral injection of 10(8) viral particles of SFV-IL-12 or SFV-enh-IL-12 induced>or=80% complete tumor regressions with long-term tumor-free survival. However, lower doses of SFV-enhIL-12 were more efficient than SFV-IL-12 in inducing antitumoral responses, indicating a positive correlation between the IL-12 expression level and the therapeutic effect. Moreover, repeated intratumoral injections of suboptimal doses of SFV-enhIL-12 increased the antitumoral response. In all cases SFV vectors were more efficient at eliminating tumors than a first-generation adenovirus vector expressing IL-12. In addition, the antitumoral effect of SFV vectors was only moderately affected by preimmunization of animals with high doses of SFV vectors. This antitumoral effect was produced, at least partially, by a potent CTL-mediated immune response.
- Generation and characterization of human iPSC line generated from mesenchymal stem cells derived from adipose tissue(Elsevier, 2016) Rodriguez-Madoz, J.R. (Juan Roberto); Rodriguez, S. (Saray); Andreu, E.J. (Enrique José); Barajas, M. (Miguel); Mazo, M. (Manuel); Prosper-Cardoso, F. (Felipe); Abizanda-Sarasa, G. (Gloria); Zapata-Linares, N.M. (Natalia María)Abstract In this work, mesenchymal stem cells derived from adipose tissue (ADSCs) were used for the generation of the human-induced pluripotent stem cell line G15.AO. Cell reprogramming was performed using retroviral vectors containing the Yamanaka factors, and the generated G15.AO hiPSC line showed normal karyotype, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation ability to the three germ layers.