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1.
Mol Ther Nucleic Acids ; 32: 322-339, 2023 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-37125150

RESUMEN

Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human T cells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 3' long terminal repeat of the LOP LVs in order to control transgene expression in human primary T cells. Importantly, inducible primary T cells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced T cells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivator-free, all-one-one system capable of generating Dox-inducible ATMPs.

2.
Nat Commun ; 11(1): 5712, 2020 11 11.
Artículo en Inglés | MEDLINE | ID: mdl-33177501

RESUMEN

Nearly half of the human genome is made of transposable elements (TEs) whose activity continues to impact its structure and function. Among them, Long INterspersed Element class 1 (LINE-1 or L1) elements are the only autonomously active TEs in humans. L1s are expressed and mobilized in different cancers, generating mutagenic insertions that could affect tumor malignancy. Tumor suppressor microRNAs are ∼22nt RNAs that post-transcriptionally regulate oncogene expression and are frequently downregulated in cancer. Here we explore whether they also influence L1 mobilization. We show that downregulation of let-7 correlates with accumulation of L1 insertions in human lung cancer. Furthermore, we demonstrate that let-7 binds to the L1 mRNA and impairs the translation of the second L1-encoded protein, ORF2p, reducing its mobilization. Overall, our data reveals that let-7, one of the most relevant microRNAs, maintains somatic genome integrity by restricting L1 retrotransposition.


Asunto(s)
Elementos de Nucleótido Esparcido Largo/genética , Neoplasias Pulmonares/genética , MicroARNs/genética , Regiones no Traducidas 3' , Adenocarcinoma del Pulmón/genética , Sitios de Unión , Carcinoma de Células Escamosas/genética , Endonucleasas/genética , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ADN Polimerasa Dirigida por ARN/genética , Células Tumorales Cultivadas , Secuenciación Completa del Genoma
3.
Philos Trans R Soc Lond B Biol Sci ; 375(1795): 20190346, 2020 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-32075559

RESUMEN

The cell culture-based retrotransposition reporter assay has been (and is) an essential tool for the study of vertebrate Long INterspersed Elements (LINEs). Developed more than 20 years ago, this assay has been instrumental in characterizing the role of LINE-encoded proteins in retrotransposition, understanding how ribonucleoprotein particles are formed, how host factors regulate LINE mobilization, etc. Moreover, variations of the conventional assay have been developed to investigate the biology of other currently active human retrotransposons, such as Alu and SVA. Here, we describe a protocol that allows combination of the conventional cell culture-based LINE-1 retrotransposition reporter assay with short interfering RNAs (siRNAs) and microRNA (miRNAs) mimics or inhibitors, which has allowed us to uncover specific miRNAs and host factors that regulate retrotransposition. The protocol described here is highly reproducible, quantitative, robust and flexible, and allows the study of several small RNA classes and various retrotransposons. To illustrate its utility, here we show that siRNAs to Fanconi anaemia proteins (FANC-A and FANC-C) and an inhibitor of miRNA-20 upregulate and downregulate human L1 retrotransposition, respectively. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.


Asunto(s)
Técnicas Genéticas , Elementos de Nucleótido Esparcido Largo , MicroARNs/genética , ARN Interferente Pequeño/genética , Retroelementos , Técnicas de Cultivo de Célula
4.
Mob DNA ; 10: 9, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30899334

RESUMEN

Human stem cells harbor significant potential for basic and clinical translational research as well as regenerative medicine. Currently ~ 3000 adult and ~ 30 pluripotent stem cell-based, interventional clinical trials are ongoing worldwide, and numbers are increasing continuously. Although stem cells are promising cell sources to treat a wide range of human diseases, there are also concerns regarding potential risks associated with their clinical use, including genomic instability and tumorigenesis concerns. Thus, a deeper understanding of the factors and molecular mechanisms contributing to stem cell genome stability are a prerequisite to harnessing their therapeutic potential for degenerative diseases. Chemical and physical factors are known to influence the stability of stem cell genomes, together with random mutations and Copy Number Variants (CNVs) that accumulated in cultured human stem cells. Here we review the activity of endogenous transposable elements (TEs) in human multipotent and pluripotent stem cells, and the consequences of their mobility for genomic integrity and host gene expression. We describe transcriptional and post-transcriptional mechanisms antagonizing the spread of TEs in the human genome, and highlight those that are more prevalent in multipotent and pluripotent stem cells. Notably, TEs do not only represent a source of mutations/CNVs in genomes, but are also often harnessed as tools to engineer the stem cell genome; thus, we also describe and discuss the most widely applied transposon-based tools and highlight the most relevant areas of their biomedical applications in stem cells. Taken together, this review will contribute to the assessment of the risk that endogenous TE activity and the application of genetically engineered TEs constitute for the biosafety of stem cells to be used for substitutive and regenerative cell therapies.

5.
Cell Chem Biol ; 26(8): 1095-1109.e14, 2019 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-31155508

RESUMEN

Retrotransposons are a type of transposable element (TE) that have amplified to astonishing numbers in mammalian genomes, comprising more than a third of the human and mouse genomes. Long interspersed element class 1 (LINE-1 or L1) retrotransposons are abundant and currently active retroelements in the human and mouse genomes. Similarly, long terminal repeat (LTR)-containing retrotransposons are abundant in both genomes, although only active in mice. LTR- and LINE-1-retroelements use different mechanisms for retrotransposition, although both involve the reverse transcription of an intermediate retroelement-derived RNA. Retrotransposon activity continues to effect the germline and somatic genomes, generating interindividual variability over evolution and potentially influencing cancer and brain physiology, respectively. However, relatively little is known about the functional consequences of retrotransposition. In this study, we have synthesized and characterized reverse transcriptase inhibitors specific for mammalian LINE-1 retrotransposons, which might help deciphering the functional impact of retrotransposition in vivo.


Asunto(s)
Didesoxinucleósidos/farmacología , Elementos de Nucleótido Esparcido Largo/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Línea Celular , Didesoxinucleósidos/síntesis química , Didesoxinucleósidos/química , Células HEK293 , Células HeLa , Humanos , Estructura Molecular , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/química
6.
Methods Mol Biol ; 1400: 1-19, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26895043

RESUMEN

Transposable elements (TEs) have been considered traditionally as junk DNA, i.e., DNA sequences that despite representing a high proportion of genomes had no evident cellular functions. However, over the last decades, it has become undeniable that not only TE-derived DNA sequences have (and had) a fundamental role during genome evolution, but also TEs have important implications in the origin and evolution of many genomic disorders. This concise review provides a brief overview of the different types of TEs that can be found in genomes, as well as a list of techniques and methods used to study their impact and mobilization. Some of these techniques will be covered in detail in this Method Book.


Asunto(s)
Elementos Transponibles de ADN , Genoma Humano , Genómica , Regulación de la Expresión Génica , Genómica/métodos , Humanos , Elementos de Nucleótido Esparcido Largo , Retroelementos
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