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Dr. karim benabdellah
Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research

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0 Gene editing
0 Crispr-Cas9
0 AAV
0 CAR T cell
0 Lentiviral Vectors

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Lentiviral Vectors
Crispr-Cas9
Gene editing
CAR T cell

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Journal article
Published: 06 August 2021 in Pharmaceutics
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Integration-deficient lentiviral vectors (IDLVs) have recently generated increasing interest, not only as a tool for transient gene delivery, but also as a technique for detecting off-target cleavage in gene-editing methodologies which rely on customized endonucleases (ENs). Despite their broad potential applications, the efficacy of IDLVs has historically been limited by low transgene expression and by the reduced sensitivity to detect low-frequency off-target events. We have previously reported that the incorporation of the chimeric sequence element IS2 into the long terminal repeat (LTR) of IDLVs increases gene expression levels, while also reducing the episome yield inside transduced cells. Our study demonstrates that the effectiveness of IDLVs relies on the balance between two parameters which can be modulated by the inclusion of IS2 sequences. In the present study, we explore new IDLV configurations harboring several elements based on IS2 modifications engineered to mediate more efficient transgene expression without affecting the targeted cell load. Of all the insulators and configurations analysed, the insertion of the IS2 into the 3′LTR produced the best results. After demonstrating a DAPI-low nuclear gene repositioning of IS2-containing episomes, we determined whether, in addition to a positive effect on transcription, the IS2 could improve the capture of IDLVs on double strand breaks (DSBs). Thus, DSBs were randomly generated, using the etoposide or locus-specific CRISPR-Cas9. Our results show that the IS2 element improved the efficacy of IDLV DSB detection. Altogether, our data indicate that the insertion of IS2 into the LTR of IDLVs improved, not only their transgene expression levels, but also their ability to be inserted into existing DSBs. This could have significant implications for the development of an unbiased detection tool for off-target cleavage sites from different specific nucleases.

ACS Style

Marina Cortijo-Gutiérrez; Sabina Sánchez-Hernández; María Tristán-Manzano; Noelia Maldonado-Pérez; Lourdes Lopez-Onieva; Pedro Real; Concha Herrera; Juan Marchal; Francisco Martin; Karim Benabdellah. Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks. Pharmaceutics 2021, 13, 1217 .

AMA Style

Marina Cortijo-Gutiérrez, Sabina Sánchez-Hernández, María Tristán-Manzano, Noelia Maldonado-Pérez, Lourdes Lopez-Onieva, Pedro Real, Concha Herrera, Juan Marchal, Francisco Martin, Karim Benabdellah. Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks. Pharmaceutics. 2021; 13 (8):1217.

Chicago/Turabian Style

Marina Cortijo-Gutiérrez; Sabina Sánchez-Hernández; María Tristán-Manzano; Noelia Maldonado-Pérez; Lourdes Lopez-Onieva; Pedro Real; Concha Herrera; Juan Marchal; Francisco Martin; Karim Benabdellah. 2021. "Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks." Pharmaceutics 13, no. 8: 1217.

Preprint content
Published: 20 March 2021
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Background Chimeric antigen receptor (CAR) T cells directed against CD19 have achieved impressive outcomes for the treatment of relapsed/refractory B lineage lymphoid neoplasms. However, CAR-T therapy still has important limitations due to severe side effects and the lack of efficiency in 40-50% of the patients. Most CARs-T products are generated using retroviral vectors with strong promoters. However, high CAR expression levels can lead to tonic signalling, premature exhaustion and over-stimulation of CAR-T cells, reducing efficacy and increasing side effects. TCR-like expression of the CAR through genome editing resulted in enhanced anti-tumour potency, reducing tonic signalling and improving CAR-T phenotype. In this manuscript, we searched for LVs that mimic the TCR expression pattern as a closer-to-clinic alternative for the generation of improved CAR-T cells. Methods Different LVs containing viral and human promoters were analysed to select those that closely mimic a TCR-like kinetic profile upon T-cell activation. WAS gene proximal promoter-driven LVs (AW-LVs) were selected to express a second generation 4-1BB aCD19 CAR (ARI-0001) into T cells to generate AWARI CAR-T cells. TCR-like AWARI and EF1α-driven ARI CAR T cells were analysed for in vitro and in vivo killing efficiency using leukaemia and lymphoma cellular models. Tonic signalling, exhaustion markers and phenotype were determined by flow cytometry. Large-scale automated manufacturing of AWARI CAR-T cells was performed in a CliniMACs Prodigy bioreactor. Results Our data showed that LVs expressing the transgene through the WAS gene proximal promoter mimic very closely the TCR (CD3) expression pattern kinetic upon TCR stimulation or antigen encounter. Compared to EF1α-driven ARI CAR-T cells, AWARI CAR-T cells exhibited a higher proportion of naïve/stem cell memory T cells with less exhausted phenotype after efficient killing of CD19+ cells both in vitro and in vivo. AWARI CAR-T cells also showed lower tonic signalling and reduced secretion of pro-inflammatory cytokines and were efficiently manufactured in large-scale GMP-like conditions. Conclusions WAS-gene-promoter driven LVs can be used to generate physiological 4-1BB-CAR-T cell products with lower tonic signalling, improved phenotype and a safer profile. We propose the use of TCR-like LVs as an alternative to strong-promoter driven LVs for the generation of CAR-T products.

ACS Style

María Tristán-Manzano; Noelia Maldonado-Pérez; Pedro Justicia-Lirio; Pilar Muñoz; Marina Cortijo-Gutiérrez; Kristina Pavlovic; Rosario Jiménez-Moreno; Sonia Nogueras; Mdolores Carmona; Sabina Sánchez-Hernández; Araceli Aguilar-González; María Castella; Manel Juan; Concepción Marañón; Karim Benabdellah; Concha Herrera; Francisco Martin. Physiological (TCR-like) regulated lentiviral vectors for the generation of improved CAR-T cells. 2021, 1 .

AMA Style

María Tristán-Manzano, Noelia Maldonado-Pérez, Pedro Justicia-Lirio, Pilar Muñoz, Marina Cortijo-Gutiérrez, Kristina Pavlovic, Rosario Jiménez-Moreno, Sonia Nogueras, Mdolores Carmona, Sabina Sánchez-Hernández, Araceli Aguilar-González, María Castella, Manel Juan, Concepción Marañón, Karim Benabdellah, Concha Herrera, Francisco Martin. Physiological (TCR-like) regulated lentiviral vectors for the generation of improved CAR-T cells. . 2021; ():1.

Chicago/Turabian Style

María Tristán-Manzano; Noelia Maldonado-Pérez; Pedro Justicia-Lirio; Pilar Muñoz; Marina Cortijo-Gutiérrez; Kristina Pavlovic; Rosario Jiménez-Moreno; Sonia Nogueras; Mdolores Carmona; Sabina Sánchez-Hernández; Araceli Aguilar-González; María Castella; Manel Juan; Concepción Marañón; Karim Benabdellah; Concha Herrera; Francisco Martin. 2021. "Physiological (TCR-like) regulated lentiviral vectors for the generation of improved CAR-T cells." , no. : 1.

Review
Published: 30 December 2020 in Cancers
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Exosomes are lipid bilayer particles released from cells into their surrounding environment. These vesicles are mediators of near and long-distance intercellular communication and affect various aspects of cell biology. In addition to their biological function, they play an increasingly important role both in diagnosis and as therapeutic agents. In this paper, we review recent literature related to the molecular composition of exosomes, paying special attention to their role in pathogenesis, along with their application as biomarkers and as therapeutic tools. In this context, we analyze the potential use of exosomes in biomedicine, as well as the limitations that preclude their wider application.

ACS Style

Houssam Aheget; Loubna Mazini; Francisco Martin; Boutaïna Belqat; Juan Antonio Marchal; Karim Benabdellah. Exosomes: Their Role in Pathogenesis, Diagnosis and Treatment of Diseases. Cancers 2020, 13, 84 .

AMA Style

Houssam Aheget, Loubna Mazini, Francisco Martin, Boutaïna Belqat, Juan Antonio Marchal, Karim Benabdellah. Exosomes: Their Role in Pathogenesis, Diagnosis and Treatment of Diseases. Cancers. 2020; 13 (1):84.

Chicago/Turabian Style

Houssam Aheget; Loubna Mazini; Francisco Martin; Boutaïna Belqat; Juan Antonio Marchal; Karim Benabdellah. 2020. "Exosomes: Their Role in Pathogenesis, Diagnosis and Treatment of Diseases." Cancers 13, no. 1: 84.

Review article
Published: 29 September 2020 in Frontiers in Immunology
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Genome editing technologies not only provide unprecedented opportunities to study basic cellular system functionality but also improve the outcomes of several clinical applications. In this review, we analyze various gene editing techniques used to fine-tune immune systems from a basic research and clinical perspective. We discuss recent advances in the development of programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases. We also discuss the use of programmable nucleases and their derivative reagents such as base editing tools to engineer immune cells via gene disruption, insertion, and rewriting of T cells and other immune components, such natural killers (NKs) and hematopoietic stem and progenitor cells (HSPCs). In addition, with regard to chimeric antigen receptors (CARs), we describe how different gene editing tools enable healthy donor cells to be used in CAR T therapy instead of autologous cells without risking graft-versus-host disease or rejection, leading to reduced adoptive cell therapy costs and instant treatment availability for patients. We pay particular attention to the delivery of therapeutic transgenes, such as CARs, to endogenous loci which prevents collateral damage and increases therapeutic effectiveness. Finally, we review creative innovations, including immune system repurposing, that facilitate safe and efficient genome surgery within the framework of clinical cancer immunotherapies.

ACS Style

Kristina Pavlovic; María Tristán-Manzano; Noelia Maldonado-Pérez; Marina Cortijo Gutiérrez; Sabina Sánchez-Hernández; Pedro Justicia-Lirio; M. Dolores Carmona; Concha Herrera; Francisco Martin; Karim Benabdellah. Using Gene Editing Approaches to Fine-Tune the Immune System. Frontiers in Immunology 2020, 11, 570672 .

AMA Style

Kristina Pavlovic, María Tristán-Manzano, Noelia Maldonado-Pérez, Marina Cortijo Gutiérrez, Sabina Sánchez-Hernández, Pedro Justicia-Lirio, M. Dolores Carmona, Concha Herrera, Francisco Martin, Karim Benabdellah. Using Gene Editing Approaches to Fine-Tune the Immune System. Frontiers in Immunology. 2020; 11 ():570672.

Chicago/Turabian Style

Kristina Pavlovic; María Tristán-Manzano; Noelia Maldonado-Pérez; Marina Cortijo Gutiérrez; Sabina Sánchez-Hernández; Pedro Justicia-Lirio; M. Dolores Carmona; Concha Herrera; Francisco Martin; Karim Benabdellah. 2020. "Using Gene Editing Approaches to Fine-Tune the Immune System." Frontiers in Immunology 11, no. : 570672.

Review
Published: 26 July 2020 in Journal of Clinical Medicine
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Summary: Exosomes are extracellular vesicles released by the vast majority of cell types both in vivo and ex vivo, upon the fusion of multivesicular bodies (MVBs) with the cellular plasma membrane. Two main functions have been attributed to exosomes: their capacity to transport proteins, lipids and nucleic acids between cells and organs, as well as their potential to act as natural intercellular communicators in normal biological processes and in pathologies. From a clinical perspective, the majority of applications use exosomes as biomarkers of disease. A new approach uses exosomes as biologically active carriers to provide a platform for the enhanced delivery of cargo in vivo. One of the major limitations in developing exosome-based therapies is the difficulty of producing sufficient amounts of safe and efficient exosomes. The identification of potential proteins involved in exosome biogenesis is expected to directly cause a deliberate increase in exosome production. In this review, we summarize the current state of knowledge regarding exosomes, with particular emphasis on their structural features, biosynthesis pathways, production techniques and potential clinical applications.

ACS Style

Houssam Aheget; María Tristán-Manzano; Loubna Mazini; Marina Cortijo-Gutierrez; Pablo Galindo-Moreno; Concha Herrera; Francisco Martin; Juan Antonio Marchal; Karim Benabdellah. Exosome: A New Player in Translational Nanomedicine. Journal of Clinical Medicine 2020, 9, 2380 .

AMA Style

Houssam Aheget, María Tristán-Manzano, Loubna Mazini, Marina Cortijo-Gutierrez, Pablo Galindo-Moreno, Concha Herrera, Francisco Martin, Juan Antonio Marchal, Karim Benabdellah. Exosome: A New Player in Translational Nanomedicine. Journal of Clinical Medicine. 2020; 9 (8):2380.

Chicago/Turabian Style

Houssam Aheget; María Tristán-Manzano; Loubna Mazini; Marina Cortijo-Gutierrez; Pablo Galindo-Moreno; Concha Herrera; Francisco Martin; Juan Antonio Marchal; Karim Benabdellah. 2020. "Exosome: A New Player in Translational Nanomedicine." Journal of Clinical Medicine 9, no. 8: 2380.

Journal article
Published: 18 June 2020 in Cells
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In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.

ACS Style

Sabina Sánchez-Hernández; Araceli Aguilar-González; Beatriz Guijarro-Albaladejo; Noelia Maldonado-Pérez; Iris Ramos-Hernández; Marina Cortijo-Gutiérrez; Rosario María Sánchez Martín; Karim Benabdellah; Francisco Martin. Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System. Cells 2020, 9, 1492 .

AMA Style

Sabina Sánchez-Hernández, Araceli Aguilar-González, Beatriz Guijarro-Albaladejo, Noelia Maldonado-Pérez, Iris Ramos-Hernández, Marina Cortijo-Gutiérrez, Rosario María Sánchez Martín, Karim Benabdellah, Francisco Martin. Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System. Cells. 2020; 9 (6):1492.

Chicago/Turabian Style

Sabina Sánchez-Hernández; Araceli Aguilar-González; Beatriz Guijarro-Albaladejo; Noelia Maldonado-Pérez; Iris Ramos-Hernández; Marina Cortijo-Gutiérrez; Rosario María Sánchez Martín; Karim Benabdellah; Francisco Martin. 2020. "Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System." Cells 9, no. 6: 1492.

Concise review
Published: 06 March 2020 in STEM CELLS Translational Medicine
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Over recent decades, gene therapy, which has enabled the treatment of several incurable diseases, has undergone a veritable revolution. Cell therapy has also seen major advances in the treatment of various diseases, particularly through the use of adult stem cells (ASCs). The combination of gene and cell therapy (GCT) has opened up new opportunities to improve advanced therapy medicinal products for the treatment of several diseases. Despite the considerable potential of GCT, the use of retroviral vectors has major limitations with regard to oncogene transactivation and the lack of physiological expression. Recently, gene therapists have focused on genome editing (GE) technologies as an alternative strategy. In this review, we discuss the potential benefits of using GE technologies to improve GCT approaches based on ASCs. We will begin with a brief summary of different GE platforms and techniques and will then focus on key therapeutic approaches that have been successfully used to treat diseases in animal models. Finally, we discuss whether ASC GE could become a real alternative to retroviral vectors in a GCT setting.

ACS Style

Karim Benabdellah; Sabina Sánchez‐Hernández; Araceli Aguilar-González; Noelia Maldonado-Pérez; Alejandra Gutierrez‐Guerrero; Marina Cortijo Gutiérrez; Iris Ramos‐Hernández; María Tristán; Pablo Galindo‐Moreno; Concha Herrera; Francisco Martin. Genome‐edited adult stem cells: Next‐generation advanced therapy medicinal products. STEM CELLS Translational Medicine 2020, 9, 674 -685.

AMA Style

Karim Benabdellah, Sabina Sánchez‐Hernández, Araceli Aguilar-González, Noelia Maldonado-Pérez, Alejandra Gutierrez‐Guerrero, Marina Cortijo Gutiérrez, Iris Ramos‐Hernández, María Tristán, Pablo Galindo‐Moreno, Concha Herrera, Francisco Martin. Genome‐edited adult stem cells: Next‐generation advanced therapy medicinal products. STEM CELLS Translational Medicine. 2020; 9 (6):674-685.

Chicago/Turabian Style

Karim Benabdellah; Sabina Sánchez‐Hernández; Araceli Aguilar-González; Noelia Maldonado-Pérez; Alejandra Gutierrez‐Guerrero; Marina Cortijo Gutiérrez; Iris Ramos‐Hernández; María Tristán; Pablo Galindo‐Moreno; Concha Herrera; Francisco Martin. 2020. "Genome‐edited adult stem cells: Next‐generation advanced therapy medicinal products." STEM CELLS Translational Medicine 9, no. 6: 674-685.

Original article
Published: 17 August 2018 in Molecular Therapy - Nucleic Acids
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Integration-defective lentiviral vectors (IDLVs) have become an important alternative tool for gene therapy applications and basic research. Unfortunately, IDLVs show lower transgene expression as compared to their integrating counterparts. In this study, we aimed to improve the expression levels of IDLVs by inserting the IS2 element, which harbors SARs and HS4 sequences, into their LTRs (SE-IS2-IDLVs). Contrary to our expectations, the presence of the IS2 element did not abrogate epigenetic silencing by histone deacetylases. In addition, the IS2 element reduced episome levels in IDLV-transduced cells. Interestingly, despite these negative effects, SE-IS2-IDLVs outperformed SE-IDLVs in terms of percentage and expression levels of the transgene in several cell lines, including neurons, neuronal progenitor cells, and induced pluripotent stem cells. We estimated that the IS2 element enhances the transcriptional activity of IDLV LTR circles 6- to 7-fold. The final effect the IS2 element in IDLVs will greatly depend on the target cell and the balance between the negative versus the positive effects of the IS2 element in each cell type. The better performance of SE-IS2-IDLVs was not due to improved stability or differences in the proportions of 1-LTR versus 2-LTR circles but probably to a re-positioning of IS2-episomes into transcriptionally active regions.

ACS Style

Sabina Sánchez-Hernández; Alejandra Gutierrez-Guerrero; Rocío Martín-Guerra; Marina Cortijo-Gutierrez; María Tristán-Manzano; Sandra Rodriguez-Perales; Laura Sanchez; Jose Luis Garcia-Perez; Jesus Chato-Astrain; Ricardo Fernandez-Valades; Ana Belén Carrillo-Galvez; Per Anderson; Rosa Montes; Pedro J. Real; Francisco Martin; Karim Benabdellah. The IS2 Element Improves Transcription Efficiency of Integration-Deficient Lentiviral Vector Episomes. Molecular Therapy - Nucleic Acids 2018, 13, 16 -28.

AMA Style

Sabina Sánchez-Hernández, Alejandra Gutierrez-Guerrero, Rocío Martín-Guerra, Marina Cortijo-Gutierrez, María Tristán-Manzano, Sandra Rodriguez-Perales, Laura Sanchez, Jose Luis Garcia-Perez, Jesus Chato-Astrain, Ricardo Fernandez-Valades, Ana Belén Carrillo-Galvez, Per Anderson, Rosa Montes, Pedro J. Real, Francisco Martin, Karim Benabdellah. The IS2 Element Improves Transcription Efficiency of Integration-Deficient Lentiviral Vector Episomes. Molecular Therapy - Nucleic Acids. 2018; 13 ():16-28.

Chicago/Turabian Style

Sabina Sánchez-Hernández; Alejandra Gutierrez-Guerrero; Rocío Martín-Guerra; Marina Cortijo-Gutierrez; María Tristán-Manzano; Sandra Rodriguez-Perales; Laura Sanchez; Jose Luis Garcia-Perez; Jesus Chato-Astrain; Ricardo Fernandez-Valades; Ana Belén Carrillo-Galvez; Per Anderson; Rosa Montes; Pedro J. Real; Francisco Martin; Karim Benabdellah. 2018. "The IS2 Element Improves Transcription Efficiency of Integration-Deficient Lentiviral Vector Episomes." Molecular Therapy - Nucleic Acids 13, no. : 16-28.

Journal article
Published: 17 November 2016 in Scientific Reports
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Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny.

ACS Style

Karim Benabdellah; Pilar Muñoz; Marién Cobo; Alejandra Gutierrez-Guerrero; Sabina Sánchez Hernández; Angélica Garcia-Perez; Per Anderson; Ana Belén Carrillo-Gálvez; Miguel G. Toscano; Francisco Martin. Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells. Scientific Reports 2016, 6, 37289 .

AMA Style

Karim Benabdellah, Pilar Muñoz, Marién Cobo, Alejandra Gutierrez-Guerrero, Sabina Sánchez Hernández, Angélica Garcia-Perez, Per Anderson, Ana Belén Carrillo-Gálvez, Miguel G. Toscano, Francisco Martin. Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells. Scientific Reports. 2016; 6 (1):37289.

Chicago/Turabian Style

Karim Benabdellah; Pilar Muñoz; Marién Cobo; Alejandra Gutierrez-Guerrero; Sabina Sánchez Hernández; Angélica Garcia-Perez; Per Anderson; Ana Belén Carrillo-Gálvez; Miguel G. Toscano; Francisco Martin. 2016. "Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells." Scientific Reports 6, no. 1: 37289.

Review
Published: 08 September 2016 in International Journal of Molecular Sciences
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The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 endonuclease (Cas9) derived from bacterial adaptive immune systems is a revolutionary tool used in both basic and applied science. It is a versatile system that enables the genome of different species to be modified by generating double strand breaks (DSBs) at specific locations. However, all of the CRISPR/Cas9 systems can also produce DSBs at off-target sites that differ substantially from on-target sites. The generation of DSBs in locations outside the intended site can produce mutations that need to be carefully monitored, especially when using these tools for therapeutic purposes. However, off-target analyses of the CRISPR/Cas9 system have been very challenging, particularly when performed directly in cells. In this manuscript, we review the different strategies developed to identify off-targets generated by CRISPR/cas9 systems and other specific nucleases (ZFNs, TALENs) in real target cells.

ACS Style

Francisco Martin; Sabina Sánchez-Hernández; Alejandra Gutiérrez-Guerrero; Javier Pinedo-Gomez; Karim Benabdellah. Biased and Unbiased Methods for the Detection of Off-Target Cleavage by CRISPR/Cas9: An Overview. International Journal of Molecular Sciences 2016, 17, 1507 .

AMA Style

Francisco Martin, Sabina Sánchez-Hernández, Alejandra Gutiérrez-Guerrero, Javier Pinedo-Gomez, Karim Benabdellah. Biased and Unbiased Methods for the Detection of Off-Target Cleavage by CRISPR/Cas9: An Overview. International Journal of Molecular Sciences. 2016; 17 (9):1507.

Chicago/Turabian Style

Francisco Martin; Sabina Sánchez-Hernández; Alejandra Gutiérrez-Guerrero; Javier Pinedo-Gomez; Karim Benabdellah. 2016. "Biased and Unbiased Methods for the Detection of Off-Target Cleavage by CRISPR/Cas9: An Overview." International Journal of Molecular Sciences 17, no. 9: 1507.

Research article
Published: 06 January 2014 in PLOS ONE
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Chromatin insulators, such as the chicken β-globin locus control region hypersensitive site 4 (HS4), and scaffold/matrix attachment regions (SARs/MARs) have been incorporated separately or in combination into retroviral vectors (RVs) in order to increase transgene expression levels, avoid silencing and reduce expression variability. However, their incorporation into RVs either produces a reduction on titer and/or expression levels or do not have sufficient effect on stem cells. In order to develop an improved insulator we decided to combine SAR elements with HS4 insulators. We designed several synthetic shorter SAR elements containing 4 or 5 MAR/SARs recognition signatures (MRS) and studied their effects on a lentiviral vector (LV) expressing eGFP through the SFFV promoter (SE). A 388 bp SAR element containing 5 MRS, named SAR2, was as efficient or superior to the other SARs analyzed. SAR2 enhanced transgene expression and reduced silencing and variability on human embryonic stem cells (hESCs). We next compared the effect of different HS4-based insulators, the HS4-Core (250 bp), the HS4-Ext (400 bp) and the HS4-650 (650 bp). All HS4 elements reduced silencing and expression variability but they also had a negative effect on transgene expression levels and titer. In general, the HS4-650 element had a better overall effect. Based on these data we developed a chimeric insulator, IS2, combining the SAR2 and the HS4-650. When incorporated into the 3′ LTR of the SE LV, the IS2 element was able to enhance expression, avoid silencing and reduce variability of expression on hESCs. Importantly, these effects were maintained after differentiation of the transduced hESCs toward the hematopoietic linage. Neither the HS4-650 nor the SAR2 elements had these effects. The IS2 element is therefore a novel insulator that confers expression stability and enhances expression of LVs on stem cells.

ACS Style

Karim Benabdellah; Alejandra Gutierrez-Guerrero; Marién Cobo; Pilar Muñoz; Francisco Martín. A Chimeric HS4-SAR Insulator (IS2) That Prevents Silencing and Enhances Expression of Lentiviral Vectors in Pluripotent Stem Cells. PLOS ONE 2014, 9, e84268 .

AMA Style

Karim Benabdellah, Alejandra Gutierrez-Guerrero, Marién Cobo, Pilar Muñoz, Francisco Martín. A Chimeric HS4-SAR Insulator (IS2) That Prevents Silencing and Enhances Expression of Lentiviral Vectors in Pluripotent Stem Cells. PLOS ONE. 2014; 9 (1):e84268.

Chicago/Turabian Style

Karim Benabdellah; Alejandra Gutierrez-Guerrero; Marién Cobo; Pilar Muñoz; Francisco Martín. 2014. "A Chimeric HS4-SAR Insulator (IS2) That Prevents Silencing and Enhances Expression of Lentiviral Vectors in Pluripotent Stem Cells." PLOS ONE 9, no. 1: e84268.

Journal article
Published: 01 May 2013 in Cell Transplantation
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Multiple sclerosis (MS) is a severe debilitating disorder characterized by progressive demyelination and axonal damage of the central nervous system (CNS). Current therapies for MS inhibit the immune response and demonstrate reasonable benefits if applied during the early phase of relapsing–remitting MS (RRMS) while there are no treatments for patients that progress neither to the chronic phase nor for the primary progressive form of the disease. In this manuscript, we have studied the therapeutic efficacy of a cell and gene therapy strategy for the treatment of a mouse model of chronic MS [myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE)]. We used allogenic mesenchymal stem cells (MSCs) as a therapeutic tool and also as vehicle to deliver fully processed 3.3-kDa vasoactive intestinal peptide (VIP) to the peripheral immune organs and to the inflamed CNS. Intraperitoneal administrations of MSCs expressing VIP stopped progression and reduced symptoms when administered at peak of disease. The improvement in clinical score correlated with diminished peripheral T-cell responses against MOG as well as lower inflammation, lower demyelination, and higher neuronal integrity in the CNS. Interestingly, neither lentiviral vectors expressing VIP nor unmodified MSCs were therapeutic when administer at the peak of disease. The increased therapeutic effect of MSCs expressing VIP over unmodified MSCs requires the immunoregulatory and neuroprotective roles of both VIP and MSCs and the ability of the MSCs to migrate to peripheral lymph organs and the inflamed CNS.

ACS Style

Marién Cobo; Per Anderson; Karim Benabdellah; Miguel G. Toscano; Pilar Muñoz; Angélica García-Pérez; Iván Gutierrez; Mario Delgado; Francisco Martin. Mesenchymal Stem Cells Expressing Vasoactive Intestinal Peptide Ameliorate Symptoms in a Model of Chronic Multiple Sclerosis. Cell Transplantation 2013, 22, 839 -854.

AMA Style

Marién Cobo, Per Anderson, Karim Benabdellah, Miguel G. Toscano, Pilar Muñoz, Angélica García-Pérez, Iván Gutierrez, Mario Delgado, Francisco Martin. Mesenchymal Stem Cells Expressing Vasoactive Intestinal Peptide Ameliorate Symptoms in a Model of Chronic Multiple Sclerosis. Cell Transplantation. 2013; 22 (5):839-854.

Chicago/Turabian Style

Marién Cobo; Per Anderson; Karim Benabdellah; Miguel G. Toscano; Pilar Muñoz; Angélica García-Pérez; Iván Gutierrez; Mario Delgado; Francisco Martin. 2013. "Mesenchymal Stem Cells Expressing Vasoactive Intestinal Peptide Ameliorate Symptoms in a Model of Chronic Multiple Sclerosis." Cell Transplantation 22, no. 5: 839-854.

Book chapter
Published: 27 February 2013 in Gene Therapy - Tools and Potential Applications
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Alejandra Gutierrez-Guerrero; Karim Benabdellah. Gene Therapy for Primary Immunodeficiencies. Gene Therapy - Tools and Potential Applications 2013, 1 .

AMA Style

Alejandra Gutierrez-Guerrero, Karim Benabdellah. Gene Therapy for Primary Immunodeficiencies. Gene Therapy - Tools and Potential Applications. 2013; ():1.

Chicago/Turabian Style

Alejandra Gutierrez-Guerrero; Karim Benabdellah. 2013. "Gene Therapy for Primary Immunodeficiencies." Gene Therapy - Tools and Potential Applications , no. : 1.

Journal article
Published: 01 January 2013 in Disease Models & Mechanisms
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Summary Mutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS), which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK) development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN) pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells) resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41α, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFβ and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed in WAS patients and as a cellular model to study new therapeutic strategies.

ACS Style

Miguel G. Toscano; Per Anderson; Pilar Muñoz; Gema Lucena; Marién Cobo; Karim Benabdellah; Philip Gregory; Michael C. Holmes; Francisco Martin. Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome. Disease Models & Mechanisms 2013, 6, 544 -554.

AMA Style

Miguel G. Toscano, Per Anderson, Pilar Muñoz, Gema Lucena, Marién Cobo, Karim Benabdellah, Philip Gregory, Michael C. Holmes, Francisco Martin. Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome. Disease Models & Mechanisms. 2013; 6 (2):544-554.

Chicago/Turabian Style

Miguel G. Toscano; Per Anderson; Pilar Muñoz; Gema Lucena; Marién Cobo; Karim Benabdellah; Philip Gregory; Michael C. Holmes; Francisco Martin. 2013. "Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome." Disease Models & Mechanisms 6, no. 2: 544-554.

Research article
Published: 14 June 2012 in PLOS ONE
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Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45−CD31+CD34+) and hematopoietic cells (CD45+). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP+ cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP+ cells showed marking of different subpopulations at different days of differentiation. At days 10–15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45−CD31+CD34dim and CD45+CD31+CD34dim) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45+CD33+). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45−CD31low/−CD34− cells, a population that disappeared at later stages of differentiation. We showed that the eGFP+CD45−CD31+ population generate 5 times more CD45+ cells than the eGFP−CD45−CD31+ indicating that the AWE vector was identifying a subpopulation inside the CD45−CD31+ cells with higher hemogenic capacity. We also showed generation of CD45+ cells from the eGFP+CD45−CD31low/−CD34− population but not from the eGFP−CD45−CD31low/−CD34− cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.

ACS Style

Pilar Muñoz; Miguel G. Toscano; Pedro Real; Karim Benabdellah; Marién Cobo; Clara Bueno; Veronica Ramos-Mejia; Pablo Menendez; Per Anderson; Francisco Martín. Specific Marking of hESCs-Derived Hematopoietic Lineage by WAS-Promoter Driven Lentiviral Vectors. PLOS ONE 2012, 7, e39091 .

AMA Style

Pilar Muñoz, Miguel G. Toscano, Pedro Real, Karim Benabdellah, Marién Cobo, Clara Bueno, Veronica Ramos-Mejia, Pablo Menendez, Per Anderson, Francisco Martín. Specific Marking of hESCs-Derived Hematopoietic Lineage by WAS-Promoter Driven Lentiviral Vectors. PLOS ONE. 2012; 7 (6):e39091.

Chicago/Turabian Style

Pilar Muñoz; Miguel G. Toscano; Pedro Real; Karim Benabdellah; Marién Cobo; Clara Bueno; Veronica Ramos-Mejia; Pablo Menendez; Per Anderson; Francisco Martín. 2012. "Specific Marking of hESCs-Derived Hematopoietic Lineage by WAS-Promoter Driven Lentiviral Vectors." PLOS ONE 7, no. 6: e39091.

Journal article
Published: 24 January 2012 in Infection, Genetics and Evolution
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Among trypanosomatids, the genus Phytomonas is the only one specifically adapted to infect plants. These hosts provide a particular habitat with a plentiful supply of carbohydrates. Phytomonas sp. lacks a cytochrome-mediated respiratory chain and Krebs cycle, and ATP production relies predominantly on glycolysis. We have characterised the complete gene encoding a putative pyruvate/indolepyruvate decarboxylase (PDC/IPDC) (548 amino acids) of P. serpens, that displays high amino acid sequence similarity with phytobacteria and Leishmania enzymes. No orthologous PDC/IPDC genes were found in Trypanosoma cruzi or T. brucei. Conservation of the PDC/IPDC gene sequence was verified in 14 Phytomonas isolates. A phylogenetic analysis shows that Phytomonas protein is robustly monophyletic with Leishmania spp. and C. fasciculata enzymes. In the trees this clade appears as a sister group of indolepyruvate decarboxylases of γ-proteobacteria. This supports the proposition that a horizontal gene transfer event from a donor phytobacteria to a recipient ancestral trypanosome has occurred prior to the separation between Phytomonas, Leishmania and Crithidia. We have measured the PDC activity in P. serpens cell extracts. The enzyme has a Km value for pyruvate of 1.4 mM. The acquisition of a PDC, a key enzyme in alcoholic fermentation, explains earlier observations that ethanol is one of the major end-products of glucose catabolism under aerobic and anaerobic conditions. This represents an alternative and necessary route to reoxidise part of the NADH produced in the highly demanding glycolytic pathway and highlights the importance of this type of event in metabolic adaptation.

ACS Style

Susan Ienne; Georgios Pappas Jr; Karim Benabdellah; Antonio González; Bianca Zingales. Horizontal gene transfer confers fermentative metabolism in the respiratory-deficient plant trypanosomatid Phytomonas serpens. Infection, Genetics and Evolution 2012, 12, 539 -548.

AMA Style

Susan Ienne, Georgios Pappas Jr, Karim Benabdellah, Antonio González, Bianca Zingales. Horizontal gene transfer confers fermentative metabolism in the respiratory-deficient plant trypanosomatid Phytomonas serpens. Infection, Genetics and Evolution. 2012; 12 (3):539-548.

Chicago/Turabian Style

Susan Ienne; Georgios Pappas Jr; Karim Benabdellah; Antonio González; Bianca Zingales. 2012. "Horizontal gene transfer confers fermentative metabolism in the respiratory-deficient plant trypanosomatid Phytomonas serpens." Infection, Genetics and Evolution 12, no. 3: 539-548.

Full paper
Published: 16 November 2011 in New Phytologist
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• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis‐associated traits, we report the first genome‐wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25 906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis‐specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis‐related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

ACS Style

E. Tisserant; A. Kohler; P. Dozolme‐Seddas; Raffaella Balestrini; Karim Benabdellah; A. Colard; Daniel Croll; C. Da Silva; S. K. Gomez; R. Koul; N. Ferrol; V. Fiorilli; D. Formey; Ph. Franken; N. Helber; Mohamed Hijri; Luisa Lanfranco; E. Lindquist; Y. Liu; M. Malbreil; E. Morin; Julie Poulain; H. Shapiro; D. van Tuinen; A. Waschke; Concepcion Azcon; G. Bécard; Paola Bonfante; M. J. Harrison; Helge Küster; P. Lammers; U. Paszkowski; N. Requena; S. A. Rensing; Christophe Roux; Ian Sanders; Yair Shachar-Hill; Gerald Tuskan; Peter Young; V. Gianinazzi‐Pearson; F. Martin. The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont. New Phytologist 2011, 193, 755 -769.

AMA Style

E. Tisserant, A. Kohler, P. Dozolme‐Seddas, Raffaella Balestrini, Karim Benabdellah, A. Colard, Daniel Croll, C. Da Silva, S. K. Gomez, R. Koul, N. Ferrol, V. Fiorilli, D. Formey, Ph. Franken, N. Helber, Mohamed Hijri, Luisa Lanfranco, E. Lindquist, Y. Liu, M. Malbreil, E. Morin, Julie Poulain, H. Shapiro, D. van Tuinen, A. Waschke, Concepcion Azcon, G. Bécard, Paola Bonfante, M. J. Harrison, Helge Küster, P. Lammers, U. Paszkowski, N. Requena, S. A. Rensing, Christophe Roux, Ian Sanders, Yair Shachar-Hill, Gerald Tuskan, Peter Young, V. Gianinazzi‐Pearson, F. Martin. The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont. New Phytologist. 2011; 193 (3):755-769.

Chicago/Turabian Style

E. Tisserant; A. Kohler; P. Dozolme‐Seddas; Raffaella Balestrini; Karim Benabdellah; A. Colard; Daniel Croll; C. Da Silva; S. K. Gomez; R. Koul; N. Ferrol; V. Fiorilli; D. Formey; Ph. Franken; N. Helber; Mohamed Hijri; Luisa Lanfranco; E. Lindquist; Y. Liu; M. Malbreil; E. Morin; Julie Poulain; H. Shapiro; D. van Tuinen; A. Waschke; Concepcion Azcon; G. Bécard; Paola Bonfante; M. J. Harrison; Helge Küster; P. Lammers; U. Paszkowski; N. Requena; S. A. Rensing; Christophe Roux; Ian Sanders; Yair Shachar-Hill; Gerald Tuskan; Peter Young; V. Gianinazzi‐Pearson; F. Martin. 2011. "The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont." New Phytologist 193, no. 3: 755-769.

Journal article
Published: 31 October 2011 in European Journal of Soil Biology
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The limited growth and nutrition of plants growing in semi-arid zones may be overcome by the inoculation with selected soil microorganisms [bacteria or arbuscular mycorrhizal fungi (AMF)]. In this article we investigate how autochthonous AM fungi and the two most abundant cultivable bacterial groups, isolated from dried degraded (B1) and non-degraded (B2) soil, affect Trifolium repens growth under water limiting conditions. On the other hand, and with attempt to characterize biochemically both isolates, we also analysed the indole acetic acid (IAA) and proline production as well as the antioxidant response of both isolates subjected to an increasing osmotic stress degree caused by Polyethylene glycol (PEG). When the bacteria were grown in axenic culture at increasing osmotic stress caused by PEG levels (from 0 to 30%) they show different osmotic response. B2 produced the highest IAA and proline amount under the strongest stress condition (30%). Similarly, under 30% PEG, B2 showed 6 times less CAT and two times more APX than B1 while SOD resulted similar in both strains. Bacterial CAT and APX activities were more sensitive than SOD to osmotic stress which is an indication of bacterial response to drought and reflect the diversity and intrinsic osmotic stress tolerance of these both bacteria. AMF or bacterial inoculated plants widely decreased stomatal conductance and increased the relative water content, both values are important for plants growing in soil with water limitation.

ACS Style

Karim Benabdellah; Younes Abbas; Mohamed Abourouh; Ricardo Aroca; Rosario Azcón. Influence of two bacterial isolates from degraded and non-degraded soils and arbuscular mycorrhizae fungi isolated from semi-arid zone on the growth of Trifolium repens under drought conditions: Mechanisms related to bacterial effectiveness. European Journal of Soil Biology 2011, 47, 303 -309.

AMA Style

Karim Benabdellah, Younes Abbas, Mohamed Abourouh, Ricardo Aroca, Rosario Azcón. Influence of two bacterial isolates from degraded and non-degraded soils and arbuscular mycorrhizae fungi isolated from semi-arid zone on the growth of Trifolium repens under drought conditions: Mechanisms related to bacterial effectiveness. European Journal of Soil Biology. 2011; 47 (5):303-309.

Chicago/Turabian Style

Karim Benabdellah; Younes Abbas; Mohamed Abourouh; Ricardo Aroca; Rosario Azcón. 2011. "Influence of two bacterial isolates from degraded and non-degraded soils and arbuscular mycorrhizae fungi isolated from semi-arid zone on the growth of Trifolium repens under drought conditions: Mechanisms related to bacterial effectiveness." European Journal of Soil Biology 47, no. 5: 303-309.

Research article
Published: 18 August 2011 in PLOS ONE
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Lentiviral vectors (LVs) are considered one of the most promising vehicles to efficiently deliver genetic information for basic research and gene therapy approaches. Combining LVs with drug-inducible expression systems should allow tight control of transgene expression with minimal side effect on relevant target cells. A new doxycycline-regulated system based on the original TetR repressor was developed in 1998 as an alternative to the TetR-VP16 chimeras (tTA and rtTA) to avoid secondary effects due to the expression of transactivator domains. However, previously described TetR-based systems required cell cloning and/or antibiotic selection of tetracycline-responsive cells in order to achieve good regulation. In the present manuscript we have constructed a dual Tet-ON system based on two lentiviral vectors, one expressing the TetR through the spleen focus forming virus (SFFV) promoter (STetR) and a second expressing eGFP through the regulatable CMV-TetO promoter (CTetOE). Using these vectors we have demonstrated that the TetR repressor, contrary to the reverse transactivator (rtTA), can be expressed in excess to bind and modulate a high number of TetO operons. We have also showed that this dual vector system can generate regulatable bulk cell lines (expressing high levels of TetR) that are able to modulate transgene expression either by varying doxycycline concentration and/or by varying the amount of CTetOE vector genomes per cell. Based on these results we have developed a new all-in-one lentiviral vector (CEST) driving the expression of TetR through the SFFV promoter and the expression of eGFP through the doxycycline-responsive CMV-TetO operon. This vector efficiently produced Tet-ON regulatable immortalized (293T) and primary (human mesenchymal stem cells and human primary fibroblasts) cells. Bulk doxycycline-responsive cell lines express high levels of the transgene with low amount of doxycycline and are phenotypically indistinct from its parental cells.

ACS Style

Karim Benabdellah; Marién Cobo; Pilar Muñoz; Miguel G. Toscano; Francisco Martin. Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines. PLOS ONE 2011, 6, e23734 .

AMA Style

Karim Benabdellah, Marién Cobo, Pilar Muñoz, Miguel G. Toscano, Francisco Martin. Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines. PLOS ONE. 2011; 6 (8):e23734.

Chicago/Turabian Style

Karim Benabdellah; Marién Cobo; Pilar Muñoz; Miguel G. Toscano; Francisco Martin. 2011. "Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines." PLOS ONE 6, no. 8: e23734.

Book chapter
Published: 22 June 2011 in Gene Therapy - Developments and Future Perspectives
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Francisco Martin; Karim Benabdellah; Marién Cobo; Pilar Muñoz; Per Anderson; Miguel G.. New Vectors for Stable and Safe Gene Modification. Gene Therapy - Developments and Future Perspectives 2011, 1 .

AMA Style

Francisco Martin, Karim Benabdellah, Marién Cobo, Pilar Muñoz, Per Anderson, Miguel G.. New Vectors for Stable and Safe Gene Modification. Gene Therapy - Developments and Future Perspectives. 2011; ():1.

Chicago/Turabian Style

Francisco Martin; Karim Benabdellah; Marién Cobo; Pilar Muñoz; Per Anderson; Miguel G.. 2011. "New Vectors for Stable and Safe Gene Modification." Gene Therapy - Developments and Future Perspectives , no. : 1.