14 results on '"Vuelta E"'
Search Results
2. Addressing Risks Derived From the Commodification of Substances of Human Origin: A European Proposal Applicable Worldwide
- Author
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Natividad Cuende, Anna Vilarrodona, Elena Vuelta, Rosario Marazuela, Concha Herrera, Sergi Querol, Jacinto Sánchez-Ibáñez, Mar Carmona, Antonio Gayá, Jaume Tort, Dolores Hernández, Beatriz Domínguez-Gil, Institut Català de la Salut, [Cuende N] Coordinación Autonómica de Trasplantes de Andalucía, Servicio Andaluz de Salud, Sevilla, Spain. [Vilarrodona A, Querol S] Banc de Sang i Teixits, Barcelona, Spain. Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. [Vuelta E] Establecimiento de Tejidos Humanos, Fundación Clínica San Francisco, León, Spain. [Marazuela R] Organización Nacional de Trasplantes, Madrid, Spain. [Herrera C] UGC de Hematología y Unidad de Terapia Celular, Hospital Universitario Reina Sofía, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Universidad de Córdoba, Córdoba, Spain, and Vall d'Hebron Barcelona Hospital Campus
- Subjects
personas::donantes de tejidos::donantes de sangre [DENOMINACIONES DE GRUPOS] ,Transplantation ,Technology, Industry, and Agriculture::Commerce::Commodification [TECHNOLOGY, INDUSTRY, AND AGRICULTURE] ,Persons::Tissue Donors::Blood Donors [NAMED GROUPS] ,Mercantilització ,Productes biològics ,Complex Mixtures::Biological Products [CHEMICALS AND DRUGS] ,Donants de sang ,mezclas complejas::productos biológicos [COMPUESTOS QUÍMICOS Y DROGAS] ,tecnología, industria y agricultura::comercio::mercantilización [TECNOLOGÍA, INDUSTRIA Y AGRICULTURA] - Abstract
Commodification; Human substances Mercantilització; Substàncies humanes Mercantilización; Sustancias humanas In view of the public consultation recently launched by the World Health Organization on Regulatory Convergence of Cell and Gene Therapy Products and the Proposal for a Regulation on substances of human origin (SoHO) repealing the European Union Directives on Blood and on Tissues and Cells, an opportunity arises to define an ethical and transparent framework of collaboration between industry and authorities responsible for SoHO-derived products, comprising medicines, medical devices, transfusion, and transplantation. The commodification of SoHO-derived medicinal products and medical devices entails important risks to the sustainability of healthcare systems and threatens the equitable access of patients to innovative therapies. It may also jeopardize the principle of altruistic donation of SoHO that is required for the treatment and survival of thousands of patients every year. This article puts forward several proposals aimed at reconciling the ethical principles of voluntary and unpaid SoHO donation and the noncommercialization of the human body with obtaining a profit that allows business activities, while ensuring high quality, safety, and efficacy standards of tissues and cells for clinical use.
- Published
- 2023
3. A Potential Effect of Circadian Rhythm in the Delivery/Therapeutic Performance of Paclitaxel-Dendrimer Nanosystems.
- Author
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Albuquerque T, Neves AR, Paul M, Biswas S, Vuelta E, García-Tuñón I, Sánchez-Martin M, Quintela T, and Costa D
- Abstract
The circadian clock controls behavior and physiology. Presently, there is clear evidence of a connection between this timing system and cancer development/progression. Moreover, circadian rhythm consideration in the therapeutic action of anticancer drugs can enhance the effectiveness of cancer therapy. Nanosized drug delivery systems (DDS) have been demonstrated to be suitable engineered platforms for drug targeted/sustained release. The investigation of the chronobiology-nanotechnology relationship, i.e., timing DDS performance according to a patient's circadian rhythm, may greatly improve cancer clinical outcomes. In the present work, we synthesized nanosystems based on an octa-arginine (R8)-modified poly(amidoamine) dendrimer conjugated with the anticancer drug paclitaxel (PTX), G4-PTX-R8, and its physicochemical properties were revealed to be appropriate for in vitro delivery. The influence of the circadian rhythm on its cellular internalization efficiency and potential therapeutic effect on human cervical cancer cells (HeLa) was studied. Cell-internalized PTX and caspase activity, as a measure of induced apoptosis, were monitored for six time points. Higher levels of PTX and caspase-3/9 were detected at T8, suggesting that the internalization of G4-PTX-R8 into HeLa cells and apoptosis are time-specific/-regulated phenomena. For a deeper understanding, the clock protein Bmal1-the main regulator of rhythmic activity, was silenced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. Bmal1 silencing was revealed to have an impact on both PTX release and caspase activity, evidencing a potential role for circadian rhythm on drug delivery/therapeutic effect mediated by G4-PTX-R8.
- Published
- 2023
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4. Addressing Risks Derived From the Commodification of Substances of Human Origin: A European Proposal Applicable Worldwide.
- Author
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Cuende N, Vilarrodona A, Vuelta E, Marazuela R, Herrera C, Querol S, Sánchez-Ibáñez J, Carmona M, Gayá A, Tort J, Hernández D, and Domínguez-Gil B
- Subjects
- Humans, Altruism, Reference Standards, Commodification, Blood Transfusion
- Abstract
In view of the public consultation recently launched by the World Health Organization on Regulatory Convergence of Cell and Gene Therapy Products and the Proposal for a Regulation on substances of human origin (SoHO) repealing the European Union Directives on Blood and on Tissues and Cells, an opportunity arises to define an ethical and transparent framework of collaboration between industry and authorities responsible for SoHO-derived products, comprising medicines, medical devices, transfusion, and transplantation. The commodification of SoHO-derived medicinal products and medical devices entails important risks to the sustainability of healthcare systems and threatens the equitable access of patients to innovative therapies. It may also jeopardize the principle of altruistic donation of SoHO that is required for the treatment and survival of thousands of patients every year. This article puts forward several proposals aimed at reconciling the ethical principles of voluntary and unpaid SoHO donation and the noncommercialization of the human body with obtaining a profit that allows business activities, while ensuring high quality, safety, and efficacy standards of tissues and cells for clinical use., Competing Interests: N.C. is member of the Governing Council of the Agencia Española de Medicamentos y Productos Sanitarios (Spanish Medicine and Medical Device Agency), pro bono appointment (as expert of recognized prestige in the field of Health Sciences, not representing any institution). The other authors declare no conflicts of interest., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)
- Published
- 2023
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5. Novel variants in GALE cause syndromic macrothrombocytopenia by disrupting glycosylation and thrombopoiesis.
- Author
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Marín-Quílez A, Di Buduo CA, Díaz-Ajenjo L, Abbonante V, Vuelta E, Soprano PM, Miguel-García C, Santos-Mínguez S, Serramito-Gómez I, Ruiz-Sala P, Peñarrubia MJ, Pardal E, Hernández-Rivas JM, González-Porras JR, García-Tuñón I, Benito R, Rivera J, Balduini A, and Bastida JM
- Subjects
- Humans, Blood Platelets metabolism, Galactose metabolism, Glycosylation, Integrin beta1 metabolism, Megakaryocytes metabolism, Thrombopoiesis genetics, Uridine Diphosphate metabolism, Thrombocytopenia genetics, Thrombocytopenia metabolism, UDPglucose 4-Epimerase genetics, UDPglucose 4-Epimerase metabolism
- Abstract
Glycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme uridine diphosphate (UDP)-galactose-4-epimerase, encoded by GALE, is involved in galactose metabolism and protein glycosylation. Here, we studied 3 patients from 2 unrelated families who showed lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice. Whole-exome sequencing revealed 4 variants that affect GALE, 3 of those previously unreported (Pedigree A, p.Lys78ValfsX32 and p.Thr150Met; Pedigree B, p.Val128Met; and p.Leu223Pro). Platelet phenotype analysis showed giant and/or grey platelets, impaired platelet aggregation, and severely reduced alpha and dense granule secretion. Enzymatic activity of the UDP-galactose-4-epimerase enzyme was severely decreased in all patients. Immunoblotting of platelet lysates revealed reduced GALE protein levels, a significant decrease in N-acetyl-lactosamine (LacNAc), showing a hypoglycosylation pattern, reduced surface expression of gylcoprotein Ibα-IX-V (GPIbα-IX-V) complex and mature β1 integrin, and increased apoptosis. In vitro studies performed with patients-derived megakaryocytes showed normal ploidy and maturation but decreased proplatelet formation because of the impaired glycosylation of the GPIbα and β1 integrin, and reduced externalization to megakaryocyte and platelet membranes. Altered distribution of filamin A and actin and delocalization of the von Willebrand factor were also shown. Overall, this study expands our knowledge of GALE-related thrombocytopenia and emphasizes the critical role of GALE in the physiological glycosylation of key proteins involved in platelet production and function., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)
- Published
- 2023
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6. CRISPR/Cas9-Directed Gene Trap Constitutes a Selection System for Corrected BCR/ABL Leukemic Cells in CML.
- Author
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Vuelta E, Ordoñez JL, Sanz DJ, Ballesteros S, Hernández-Rivas JM, Méndez-Sánchez L, Sánchez-Martín M, and García-Tuñón I
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- Apoptosis genetics, CRISPR-Cas Systems genetics, Cell Proliferation genetics, Chronic Disease, Humans, Fusion Proteins, bcr-abl genetics, Fusion Proteins, bcr-abl metabolism, Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy, Leukemia, Myelogenous, Chronic, BCR-ABL Positive therapy
- Abstract
Chronic myeloid leukaemia (CML) is a haematological neoplasm driven by the BCR/ABL fusion oncogene. The monogenic aspect of the disease and the feasibility of ex vivo therapies in haematological disorders make CML an excellent candidate for gene therapy strategies. The ability to abolish any coding sequence by CRISPR-Cas9 nucleases offers a powerful therapeutic opportunity to CML patients. However, a definitive cure can only be achieved when only CRISPR-edited cells are selected. A gene-trapping approach combined with CRISPR technology would be an ideal approach to ensure this. Here, we developed a CRISPR-Trap strategy that efficiently inserts a donor gene trap (SA-CMV-Venus) cassette into the BCR/ABL -specific fusion point in the CML K562 human cell line. The trapping cassette interrupts the oncogene coding sequence and expresses a reporter gene that enables the selection of edited cells. Quantitative mRNA expression analyses showed significantly higher level of expression of the BCR/Venus allele coupled with a drastically lower level of BCR/ABL expression in Venus + cell fractions. Functional in vitro experiments showed cell proliferation arrest and apoptosis in selected Venus + cells. Finally, xenograft experiments with the selected Venus + cells showed a large reduction in tumour growth, thereby demonstrating a therapeutic benefit in vivo. This study represents proof of concept for the therapeutic potential of a CRISPR-Trap system as a novel strategy for gene elimination in haematological neoplasms.
- Published
- 2022
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7. A novel nonsense variant in TPM4 caused dominant macrothrombocytopenia, mild bleeding tendency and disrupted cytoskeleton remodeling.
- Author
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Marín-Quílez A, Vuelta E, Díaz-Ajenjo L, Fernández-Infante C, García-Tuñón I, Benito R, Palma-Barqueros V, Hernández-Rivas JM, González-Porras JR, Rivera J, and Bastida JM
- Subjects
- Blood Platelets metabolism, Cytoskeleton metabolism, Hemorrhage, Humans, Thrombopoiesis genetics, Tropomyosin genetics, Tropomyosin metabolism, Blood Platelet Disorders genetics, Thrombocytopenia
- Abstract
Background: Rare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants., Objectives: To characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology., Patients/methods: Index case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH-BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole-exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting., Results: Five family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin-4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP-6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up-regulation of TPM2 and TPM3 mRNA levels., Conclusions: This study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4-related thrombocytopenia., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)
- Published
- 2022
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8. Characterization of the Platelet Phenotype Caused by a Germline RUNX1 Variant in a CRISPR/Cas9-Generated Murine Model.
- Author
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Marín-Quílez A, García-Tuñón I, Fernández-Infante C, Hernández-Cano L, Palma-Barqueros V, Vuelta E, Sánchez-Martín M, González-Porras JR, Guerrero C, Benito R, Rivera J, Hernández-Rivas JM, and Bastida JM
- Subjects
- Animals, Blood Platelet Disorders blood, CRISPR-Associated Protein 9 metabolism, Core Binding Factor Alpha 2 Subunit blood, Cytoplasmic Granules genetics, Cytoplasmic Granules metabolism, Disease Models, Animal, Gene Knock-In Techniques, Genetic Predisposition to Disease, Hemostasis, Mice, Inbred C57BL, Mice, Transgenic, Phenotype, Platelet Glycoprotein GPIIb-IIIa Complex metabolism, Secretory Pathway, Thrombopoiesis, Mice, Blood Platelet Disorders genetics, Blood Platelets metabolism, CRISPR-Associated Protein 9 genetics, CRISPR-Cas Systems, Core Binding Factor Alpha 2 Subunit genetics, Mutation, Platelet Activation genetics
- Abstract
RUNX1 -related disorder ( RUNX1 -RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1 -RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1
WT/WT , RUNX1WT/L43S , and RUNX1L43S/L43S ). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1L43S/L43S and RUNX1WT/L43S mice had a significantly longer tail-bleeding time than RUNX1WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1L43S/L43S and RUNX1WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin αIIb β3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1L43S/L43S and RUNX1WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1L43S/L43S and RUNX1WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin αIIb β3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting., Competing Interests: None declared., (Thieme. All rights reserved.)- Published
- 2021
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9. CRISPR-Cas9 Technology as a Tool to Target Gene Drivers in Cancer: Proof of Concept and New Opportunities to Treat Chronic Myeloid Leukemia.
- Author
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Vuelta E, Ordoñez JL, Alonso-Pérez V, Méndez L, Hernández-Carabias P, Saldaña R, Sevilla J, Sebastián E, Muntión S, Sánchez-Guijo F, Hernández-Rivas JM, García-Tuñón I, and Sánchez-Martín M
- Subjects
- Animals, Cell Line, Tumor, Disease Models, Animal, Fusion Proteins, bcr-abl genetics, Gene Expression, Gene Targeting methods, Gene Transfer Techniques, Hematopoiesis genetics, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism, Heterografts, Humans, Leukemia, Myelogenous, Chronic, BCR-ABL Positive therapy, Mice, Neoplastic Stem Cells metabolism, Proof of Concept Study, CRISPR-Cas Systems, Gene Editing, Genetic Therapy methods, Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics, Oncogenes
- Abstract
Chronic myeloid leukemia (CML) is a hematopoietic malignancy produced by a unique oncogenic event involving the constitutively active tyrosine-kinase (TK) BCR/ABL1 . TK inhibitors (TKI) changed its prognosis and natural history. Unfortunately, ABL1 remains unaffected by TKIs. Leukemic stem cells (LSCs) remain, and resistant mutations arise during treatment. To address this problem, we have designed a therapeutic CRISPR-Cas9 deletion system targeting BCR/ABL1 . The system was efficiently electroporated to cell lines, LSCs from a CML murine model, and LSCs from CML patients at diagnosis, generating a specific ABL1 null mutation at high efficiency and allowing the edited leukemic cells to be detected and tracked. The CRISPR-Cas9 deletion system triggered cell proliferation arrest and apoptosis in murine and human CML cell lines. Patient and murine-derived xenografts with CRISPR-edited LSCs in NOD SCID gamma niches revealed that normal multipotency and repopulation ability of CRISPR edited LSCs were fully restored. Normal hematopoiesis was restored, avoiding myeloid bias. To the best of our knowledge, we show for the first time how a CRISPR-Cas9 deletion system efficiently interrupts BCR/ABL1 oncogene in primary LSCs to bestow a therapeutic benefit. This study is a proof of concept for genome editing in all those diseases, like CML, sustained by a single oncogenic event, opening up new therapeutic opportunities.
- Published
- 2021
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10. Stroma-Mediated Resistance to S63845 and Venetoclax through MCL-1 and BCL-2 Expression Changes Induced by miR-193b-3p and miR-21-5p Dysregulation in Multiple Myeloma.
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Algarín EM, Quwaider D, Campos-Laborie FJ, Díaz-Tejedor A, Mogollón P, Vuelta E, Martín-Sánchez M, San-Segundo L, González-Méndez L, Gutiérrez NC, García-Sanz R, Paíno T, De Las Rivas J, Ocio EM, and Garayoa M
- Subjects
- Antineoplastic Agents pharmacology, Bridged Bicyclo Compounds, Heterocyclic pharmacology, Drug Resistance, Neoplasm drug effects, Humans, Multiple Myeloma pathology, Pyrimidines pharmacology, Sulfonamides pharmacology, Thiophenes pharmacology, Antineoplastic Agents therapeutic use, Bridged Bicyclo Compounds, Heterocyclic therapeutic use, Multiple Myeloma drug therapy, Proto-Oncogene Proteins c-bcl-2 metabolism, Pyrimidines therapeutic use, Sulfonamides therapeutic use, Thiophenes therapeutic use
- Abstract
BH3-mimetics targeting anti-apoptotic proteins such as MCL-1 (S63845) or BCL-2 (venetoclax) are currently being evaluated as effective therapies for the treatment of multiple myeloma (MM). Interleukin 6, produced by mesenchymal stromal cells (MSCs), has been shown to modify the expression of anti-apoptotic proteins and their interaction with the pro-apoptotic BIM protein in MM cells. In this study, we assess the efficacy of S63845 and venetoclax in MM cells in direct co-culture with MSCs derived from MM patients (pMSCs) to identify additional mechanisms involved in the stroma-induced resistance to these agents. MicroRNAs miR-193b-3p and miR-21-5p emerged among the top deregulated miRNAs in myeloma cells when directly co-cultured with pMSCs, and we show their contribution to changes in MCL-1 and BCL-2 protein expression and in the activity of S63845 and venetoclax. Additionally, direct contact with pMSCs under S63845 and/or venetoclax treatment modifies myeloma cell dependence on different BCL-2 family anti-apoptotic proteins in relation to BIM, making myeloma cells more dependent on the non-targeted anti-apoptotic protein or BCL-X
L . Finally, we show a potent effect of the combination of S63845 and venetoclax even in the presence of pMSCs, which supports this combinatorial approach for the treatment of MM.- Published
- 2021
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11. Future Approaches for Treating Chronic Myeloid Leukemia: CRISPR Therapy.
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Vuelta E, García-Tuñón I, Hernández-Carabias P, Méndez L, and Sánchez-Martín M
- Abstract
The constitutively active tyrosine-kinase BCR/ABL1 oncogene plays a key role in human chronic myeloid leukemia development and disease maintenance, and determines most of the features of this leukemia. For this reason, tyrosine-kinase inhibitors are the first-line treatment, offering most patients a life expectancy like that of an equivalent healthy person. However, since the oncogene stays intact, lifelong oral medication is essential, even though this triggers adverse effects in many patients. Furthermore, leukemic stem cells remain quiescent and resistance is observed in approximately 25% of patients. Thus, new therapeutic alternatives are still needed. In this scenario, the interruption/deletion of the oncogenic sequence might be an effective therapeutic option. The emergence of CRISPR (clustered regularly interspaced short palindromic repeats) technology can offer a definitive treatment based on its capacity to induce a specific DNA double strand break. Besides, it has the advantage of providing complete and permanent oncogene knockout, while tyrosine kinase inhibitors (TKIs) only ensure that BCR-ABL1 oncoprotein is inactivated during treatment. CRISPR/Cas9 cuts DNA in a sequence-specific manner making it possible to turn oncogenes off in a way that was not previously feasible in humans. This review describes chronic myeloid leukemia (CML) disease and the main advances in the genome-editing field by which it may be treated in the future.
- Published
- 2021
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12. Establishment of a conditional Nomo1 mouse model by CRISPR/Cas9 technology.
- Author
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García-Tuñón I, Vuelta E, Lozano L, Herrero M, Méndez L, Palomero-Hernandez J, Pérez-Caro M, Pérez-García J, González-Sarmiento R, and Sánchez-Martín M
- Subjects
- Alleles, Animals, Base Sequence, CRISPR-Associated Protein 9 metabolism, Disease Models, Animal, Exons genetics, Integrases metabolism, Membrane Proteins metabolism, Mice, Inbred C57BL, Mice, Transgenic, Mosaicism, Mutation genetics, Nodal Protein metabolism, RNA, Guide, CRISPR-Cas Systems metabolism, Clustered Regularly Interspaced Short Palindromic Repeats genetics, Membrane Proteins genetics, Nodal Protein genetics
- Abstract
The Nomo1 gene mediates a wide range of biological processes of importance in embryonic development. Accordingly, constitutive perturbation of Nomo1 function may result in myriad developmental defects that trigger embryonic lethality. To extend our understanding of Nomo1 function in postnatal stages and in a tissue-specific manner, we generated a conditional knockout mouse model of Nomo1. To achieve this, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology in C57Bl/6J mouse zygotes to generate a new mouse model in which exon 3 of the Nomo1 gene is specifically flanked (or floxed) by LoxP sites (Nomo1
f/f ). Nomo1f/f mouse embryonic fibroblasts were transduced with a Cre adenovirus and efficiently recombined between LoxP sites. Genomic and expression studies in Nomo1-transduced MEFs demonstrated that the Nomo1 exon 3 is ablated. Western blot assay showed that no protein or early truncated protein is produced. In vivo assay crossing Nomo1f/f mouse with a Msi1-CRE transgenic mouse corroborated the previous findings and it showed Nomo1 exon 3 deletion at msi1+ cell compartment. This short technical report demonstrates that CRISPR/Cas9 technology is a simple and easy method for creating conditional mouse models. The Nomo1f/f mouse will be useful to researchers who wish to explore the role of Nomo1 in any developmental stage or in a tissue-specific manner.- Published
- 2020
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13. Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency.
- Author
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García-Tuñón I, Alonso-Pérez V, Vuelta E, Pérez-Ramos S, Herrero M, Méndez L, Hernández-Sánchez JM, Martín-Izquierdo M, Saldaña R, Sevilla J, Sánchez-Guijo F, Hernández-Rivas JM, and Sánchez-Martín M
- Subjects
- Alleles, Animals, Ataxia Telangiectasia Mutated Proteins genetics, Cell Line, Exons, Gene Editing methods, Humans, K562 Cells, Mice, Monophenol Monooxygenase genetics, Proto-Oncogene Proteins c-abl genetics, CRISPR-Cas Systems, Gene Knockout Techniques methods, RNA Splice Sites genetics, RNA, Guide, CRISPR-Cas Systems genetics
- Abstract
CRISPR/Cas9 allows the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
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14. Paternal exposure to environmental 17-alpha-ethinylestradiol concentrations modifies testicular transcription, affecting the sperm transcript content and the offspring performance in zebrafish.
- Author
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Valcarce DG, Vuelta E, Robles V, and Herráez MP
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- Animals, Embryo, Nonmammalian drug effects, Embryo, Nonmammalian metabolism, Female, Gene Expression, Male, Motor Activity, Paternal Exposure adverse effects, Receptors, Estrogen genetics, Receptors, Estrogen metabolism, Spermatogenesis, Transcription, Genetic drug effects, Up-Regulation, Zebrafish abnormalities, Zebrafish genetics, Zebrafish Proteins genetics, Zebrafish Proteins metabolism, Endocrine Disruptors toxicity, Ethinyl Estradiol toxicity, Spermatozoa metabolism, Testis metabolism, Water Pollutants, Chemical toxicity, Zebrafish physiology
- Abstract
The synthetic estrogen 17-α-ethinylestradiol (EE2), a major constituent in contraceptive pills, is an endocrine disrupting chemical (EDC) present in the aquatic environment at concentrations of ng/L. Developmental exposure to these low concentrations in fish can induce several disorders. Zebrafish (Danio rerio) is a perfect organism for monitoring the effects of environmental contaminants. Our hypothesis is that changes promoted by EE2 in the germ line of male adults could be transmitted to the unexposed progeny. We exposed male zebrafish to 2.5, 5 and 10ng/L of EE2 during spermatogenesis and mated them with untreated females. Detailed progeny development was studied concentrating to survival, hatching and malformations. Due to the high incidence of lymphedemas within larvae, we performed qPCR analysis of genes involved in lymphatic development (vegfc and vegfr3) and endothelial cell migration guidance (cxcr4a and cxcl12b). Estrogen receptor (ER) transcript presence was also evaluated in sperm, testis and embryos. Progenies showed a range of disorders although at a low incidence: skeletal distortions, uninflated swimbladder, lymphedema formation, cartilage deformities and otolith tethering. Swimming evaluation revealed less active locomotion. All these processes are related to pathways involving ERs (esr1, esr2a and esr2b). mRNA analysis revealed that environmental EE2 causes the up-regulation of esr1 an esr2b in testis and the increase of esr2b transcripts in sperm pointing to a link between lymphedema in embryos and ER expression impairment. We demonstrate that the effects induced by environmental toxicants can be paternally inherited and point to the changes on the sperm transcriptome as the responsible mechanism., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
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