Martínez-Turrillas, R. (Rebeca)

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    Elevated synaptic vesicle release probability in synaptophysin/gyrin family quadruple knockouts
    (eLife Sciences Publications, Ltd, 2019) Preobraschenski, J. (Julia); Martínez-Turrillas, R. (Rebeca); Raja, M.K. (Mathan K.); Wesseling, J.F. (John F.); Jahn, R. (Reinhard); Perez-Otaño, I. (Isabel); Olmo-Cabrera, S. (Sergio) del
    Synaptophysins 1 and 2 and synaptogyrins 1 and 3 constitute a major family of synaptic vesicle membrane proteins. Unlike other widely expressed synaptic vesicle proteins such as vSNAREs and synaptotagmins, the primary function has not been resolved. Here, we report robust elevation in the probability of release of readily releasable vesicles with both high and low release probabilities at a variety of synapse types from knockout mice missing all four family members. Neither the number of readily releasable vesicles, nor the timing of recruitment to the readily releasable pool was affected. The results suggest that family members serve as negative regulators of neurotransmission, acting directly at the level of exocytosis to dampen connection strength selectively when presynaptic action potentials fire at low frequency. The widespread expression suggests that chemical synapses may play a frequency filtering role in biological computation that is more elemental than presently envisioned.
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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.
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    Assessment of a New ROS1 Immunohistochemistry Clone (SP384) for the Identification of ROS1 Rearrangements in Patients with Non–Small Cell Lung Carcinoma: the ROSING Study
    (Elsevier BV, 2019) Gonzalez-Larriba, J.L. (José Luis); Plaza, M.L. (María Luz); Rodríguez-Abreu, D. (Delvys); Domine, M. (Manuel); Lozano, M.D. (María Dolores); García-González, J (Jorge); Enguita, A.B. (Ana Belén); Gomez-Roman, J. (Javier); Lázaro-Quintela, M. (Martín); Aguiar, D. (David); Saiz, M. (Mónica); Felip, E. (Enriqueta); Muriel, A. (Alfonso); López-Brea, M. (Marta); Mancheño, N. (Nuria); Arriola, E. (Edurne); Conde, E. (Esther); Garrido, P. (Pilar); Atienza-Cuevas, L. (Lidia); González-Piñeiro, A. (Ana); Jiménez, B. (Beatriz); Gurpide, A. (Alfonso); Martínez-Turrillas, R. (Rebeca); Arriola-Arellano, E. (Esperanza); Esteban-Rodríguez, I. (Isabel); Teixidó, C. (Cristina); Company, A. (Amparo); Aranda, I. (Ignacio); Hernández, S. (Susana); Moran, T. (Teresa); Castro, J. (Javier) de; Camacho, C. (Carmen); Benito, A. (Amparo); Álvarez, R. (Ramiro); Paz-Ares, L. (Luis); Mate, J.L. (Jose Luis); Sansano, I. (Irene); Reguart, N. (Noemi); López-Ríos, F. (Fernando); Artal, Á. (Ángel); Angulo, B. (Bárbara); Juan, O. (Oscar); García, F. (Felip); Abdulkader, I. (Ihab); Salido, M. (Marta); Sanz, J. (Julián); Collazo-Lorduy, A. (Ana); Rojo-Todo, F. (Federico); Insa, A. (Amelia); Pijuan, L. (Lara); Massuti, B. (Bartomeu); Azcona, E. (Eider)
    Introduction: The ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data. Methods: A total of 43 ROS1 FISH-positive and 193 ROS1 FISH-negative NSCLC samples were studied. All specimens were screened by using two antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 from Ventana Medical Systems), and the different interpretation criteria were compared with break-apart FISH (Vysis). FISH-positive samples were also analyzed with next-generation sequencing (Oncomine Dx Target Test Panel, Thermo Fisher Scientific). Results: An H-score of 150 or higher or the presence of at least 70% of tumor cells with an intensity of staining of 2+ or higher by the SP384 clone was the optimal cutoff value (both with 93% sensitivity and 100% specificity). The D4D6 clone showed similar results, with an H-score of at least 100 (91% sensitivity and 100% specificity). ROS1 expression in normal lung was more frequent with use of the SP384 clone (p < 0.0001). The ezrin gene (EZR)-ROS1 variant was associated with membranous staining and an isolated green signal FISH pattern (p = 0.001 and p = 0.017, respectively). Conclusions: The new SP384 ROS1 IHC clone showed excellent sensitivity without compromising specificity, so it is another excellent analytical option for the proposed testing algorithm.
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    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.
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    Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia
    (2023) Rodriguez-Marquez, P. (Paula); Rodriguez-Madoz, J.R. (Juan Roberto); Abizanda-Sarasa, G. (Gloria María); Illarramendi, J. (Jorge); San-Martín-Uriz, P. (Patxi); Lopez-Diaz-de-Cerio, A. (Ascensión); Rifon, J. J. (Jose J.); Villar-Fernández, S. (Sara); Viguria, M.C. (María C.); Jauregui-Jiménez, P. (Patricia); Martin-Mallo, A. (Angel); Alfonso-Piérola, A. (Ana); Inoges, S. (Susana); Rodríguez-Díaz, S. (Saray); Martínez-Turrillas, R. (Rebeca); Calleja-Cervantes, M.E. (María Erendira); Ceballos, C. (Candela); Hernaez, M. (Mikel); Redondo, M. (M.); Iglesias, E. (Elena); Calviño-Sampedro, C. (Cristina); Prosper-Cardoso, F. (Felipe); Lasarte, J.J. (Juan José)
    Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-IKO/TCRKO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.