Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells

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 30 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 transactivatorfree, all-one-one system capable of generating Dox-inducible ATMPs.Spanish ISCIII Health Research FundEuropean Union (EU) PI18/00337 PI21/00298 RD21/0017/0004 PI18/00330 PI17/00672Red TerAvJunta de Andalucia FEDER/European Cohesion Fund (FSE) for AndalusiaSpanish Government PI18/00337 PI21/00298European Union-NextGenerationEU - Maria Zambrano Senior Program RD21/0017/0004 PI18/00330 PI17/00672Ministry of Health 2016000073332-TRA PI-57069 CARTPI-0001-201 PE-CART-0031-2020 PI-0014-2016 PECART-0027-2020 ProyExcel_00875 PEER-0286-2019European Cooperation in Science and Technology (COST) 00123009/SNEO-20191072MINECO - European Regional Development Fund PLEC2021-008094Spanish Government 0006/2018FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades CA21113Spanish Government SAF2015-71589-PMCI RYC-2016-21395German Research Foundation (DFG) PY20_00619 y A-CTS-28_UGR20Biomedicine Program of the University of Granada (Spain) FPU16/05467 FPU17/02268 FPU17/04327 DIN2018-010180 DIN2020-011550 PEJ-2018-001760-

Regulation of Transposable Elements by microRNAs in Cancer

Los elementos móviles o transponibles (TEs) constituyen casi la mitad del genoma humano, y siguen afectando su estructura y función. Los únicos TEs activos y autónomos en la actualidad se denominan ‘Long INterspersed Elements class 1’ (LINE-1s or L1s), cuyas 500,000 copias forman en torno al 17% de nuestro genoma. Sólo 80-100 de estas copias siguen siendo activas, y se denominan L1s retrocompetentes o RC-L1s. Los RC-L1s tienen una longitud de 6 kb y codifican dos proteínas imprescindibles para su movilización: L1-ORF1p, una proteína de unión a ARN con actividad chaperona, y L1-ORF2p, con actividades endonucleasa y reverso transcriptasa. Los L1s se movilizan por un mecanismo de copia y pega, generando nuevas inserciones mediante la transcripción inversa de un RNA intermediario. Por el momento, se han descrito más de un centenar de inserciones causantes de enfermedad. En conjunto, esta Tesis contribuye a los campos de los elementos transponibles y de miARNs, así como a la biología del cáncer. De hecho, hemos descubierto una nueva función de la familia de miARNs supresores de tumores let-7: mantener la integridad del genoma somático controlando la retrotransposición de L1.Transposable elements (TEs) comprise nearly half of the human genome, and their ongoing activity continues to impact its structure and function. The only TEs that remain autonomously active are Long INterspersed Elements class 1 (LINE-1s or L1s) retrotransposons, whose 500,000 copies account for ~17% of our genome. Only 80-100 L1s retain the ability to mobilize, and are therefore called Retrotransposition-Competent L1s (RC-L1s). RC-L1s are 6kb long and encode two proteins that are required for their mobilization: L1-ORF1p, an RNA binding protein with chaperone activity, and L1-ORF2p, with endonuclease and reverse transcriptase activities. RC-L1s mobilize via a copy-and-paste mechanism, generating new insertions by reverse transcription of an intermediate RNA. At present, over one hundred disease-causing insertions have been reported. Altogether, this Thesis represents a contribution to the fields of mobile genetic elements and miRNAs, as well as to cancer biology. In fact, we uncover a new role for the tumor suppressor miRNA let-7 family: maintaining somatic genome integrity by restricting L1 retrotransposition.Tesis Univ. Granada

Genome-edited adult stem cells: Next-generation advanced therapy medicinal products.

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

El debate permanente

El libro trata sobre uno de los interrogantes científicos más importantes para el conocimiento de la historia de la humanidad: ¿cómo surgen, se organizan, se mueven y se transforman las sociedades? Pregunta que, al igual que los problemas centrales de la ciencia, no encuentra su origen en los estrechos muros de los ámbitos académicos, sino que responde a la necesidad que, como sociedad, tenemos de conocernos, dilucidar quiénes somos y hacia dónde vamos. De te fabula narratur: de ti, de vos, de mí, de él, de ella, de todos nosotros, habla la Historia. A través de veinte capítulos, ofrecemos al lector una aproximación a los más recientes debates, así como a flamantes investigaciones, dedicadas a la cuestión del vínculo entre los sistemas productivos americanos, las revoluciones y las transiciones sociales. Esperamos que este libro contribuya a divulgar la necesidad de conocer el pasado para comprender el presente y transformar el futuro, ofreciendo más pruebas de que la Historia habla de nosotros, aunque refiera, ocasionalmente, a indígenas, mitayos u obreras fabriles. De te fabula narratur

Subcutaneous anti-COVID-19 hyperimmune immunoglobulin for prevention of disease in asymptomatic individuals with SARS-CoV-2 infection: a double-blind, placebo-controlled, randomised clinical trialResearch in context

Summary: Background: Anti-COVID-19 hyperimmune immunoglobulin (hIG) can provide standardized and controlled antibody content. Data from controlled clinical trials using hIG for the prevention or treatment of COVID-19 outpatients have not been reported. We assessed the safety and efficacy of subcutaneous anti-COVID-19 hyperimmune immunoglobulin 20% (C19-IG20%) compared to placebo in preventing development of symptomatic COVID-19 in asymptomatic individuals with SARS-CoV-2 infection. Methods: We did a multicentre, randomized, double-blind, placebo-controlled trial, in asymptomatic unvaccinated adults (≥18 years of age) with confirmed SARS-CoV-2 infection within 5 days between April 28 and December 27, 2021. Participants were randomly assigned (1:1:1) to receive a blinded subcutaneous infusion of 10 mL with 1 g or 2 g of C19-IG20%, or an equivalent volume of saline as placebo. The primary endpoint was the proportion of participants who remained asymptomatic through day 14 after infusion. Secondary endpoints included the proportion of individuals who required oxygen supplementation, any medically attended visit, hospitalisation, or ICU, and viral load reduction and viral clearance in nasopharyngeal swabs. Safety was assessed as the proportion of patients with adverse events. The trial was terminated early due to a lack of potential benefit in the target population in a planned interim analysis conducted in December 2021. ClinicalTrials.gov registry: NCT04847141. Findings: 461 individuals (mean age 39.6 years [SD 12.8]) were randomized and received the intervention within a mean of 3.1 (SD 1.27) days from a positive SARS-CoV-2 test. In the prespecified modified intention-to-treat analysis that included only participants who received a subcutaneous infusion, the primary outcome occurred in 59.9% (91/152) of participants receiving 1 g C19-IG20%, 64.7% (99/153) receiving 2 g, and 63.5% (99/156) receiving placebo (difference in proportions 1 g C19-IG20% vs. placebo, −3.6%; 95% CI -14.6% to 7.3%, p = 0.53; 2 g C19-IG20% vs placebo, 1.1%; −9.6% to 11.9%, p = 0.85). None of the secondary clinical efficacy endpoints or virological endpoints were significantly different between study groups. Adverse event rate was similar between groups, and no severe or life-threatening adverse events related to investigational product infusion were reported. Interpretation: Our findings suggested that administration of subcutaneous human hyperimmune immunoglobulin C19-IG20% to asymptomatic individuals with SARS-CoV-2 infection was safe but did not prevent development of symptomatic COVID-19. Funding: Grifols