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Soluble receptor; Multiple sclerosis Receptor soluble; Esclerosis múltiple Receptor soluble; Esclerosi múltiple The pathogenic role of the interleukin 21 (IL-21) in different autoimmune diseases, such as multiple sclerosis (MS), has been extensively studied. However, its pleiotropic nature makes it a cytokine that may exhibit different activity depending on the immunological stage of the disease. In this study, we developed a gene therapy strategy to block the interaction between IL-21 and its receptor (IL-21R) by using adeno-associated vectors (AAV) encoding a new soluble cytokine receptor (sIL21R) protein. We tested this strategy in a murine model of experimental autoimmune encephalomyelitis (EAE), obtaining different clinical effects depending on the time at which the treatment was applied. Although the administration of the treatment during the development of the immune response was counterproductive, the preventive administration of the therapeutic vectors showed a protective effect by reducing the number of animals that developed the disease, as well as an improvement at the histopathological level and a modification of the immunological profile of the animals treated with the AAV8.sIL21R. The beneficial effect of the treatment was also observed when inducing the expression of the therapeutic molecule once the first neurological signs were established in a therapeutic approach with a doxycyline (Dox)-inducible expression system. All these clinical results highlight the pleiotropicity of this cytokine in the different clinical stages and its key role in the EAE immunopathogenesis. Open Access Funding provided by Universitat Autonoma de Barcelona. This project was supported by the Ministerio Ciencia Innovación, retos Sociedad (PI15/0271). A.E was a recipient of a VHIR fellowship.

Dissertação para obtenção do grau de Mestre no Instituto Universitário Egas Moniz A doença de MachadoJoseph (DMJ) ou ataxia espinocerebelar 3 (SCA3), é uma doença neurodegenerativa, pertencente ao grupo das doenças de poliglutamina (poliQ). Molecularmente é causada pela expansão de uma repetição CAG na região codificante do gene ATXN3. Esta é a ataxia mais comum herdada de forma dominante. Relativamente a Portugal, este é um país com elevada prevalência desta doença. No continente representam 56 % de todas as ataxias AD, correspondendo a uma prevalência de 3,1/100000 habitantes, e de 41,6/1000000 habitantes na região dos Açores. Carateriza-se pelo seu aparecimento tardio, entre os 35 e os 40 anos, e pela perda progressiva das funções motoras, levando à morte. Apesar da causa monogénica da doença, a sua fisiopatologia permanece indefinida. A mutação resulta numa proteína dobrada de forma atípica que agrega e afeta várias funções celulares, incluindo a transcrição do RNA, a homeostase da proteína e a transmissão sináptica. Atualmente, não há cura para a DMJ, no entanto, a crescente compreensão da patogénese da doença oferece diferentes caminhos para potenciais terapêuticas. Esta dissertação tem como objetivo realizar uma revisão narrativa sobre a abordagem terapêutica e terapia genética na Doença de MachadoJoseph (DMJ). Numerosos estudos têm-se focado no desenvolvimento de tratamentos que visam a doença em diferentes estágios, prevenindo a agregação de proteínas, aumentando a depuração de proteínas mutantes e combatendo os mecanismos de disfunção celular. Nos últimos anos, também tem havido um foco crescente no desenvolvimento de terapias genéticas, sugerindo que, no futuro, essa doença debilitante possa ser prevenida. MachadoJoseph disease (MJD) or spinocerebellar ataxia 3 (SCA3) is a neurodegenerative disease belonging to the group of polyglutamine (polyQ) diseases. Molecularly CA G is the expansion of a coding region of the ATXN3 gene. This is the most common dominantly inherited ataxia. Regarding Portugal, this is a country with prevalence of this disease. On the mainland 56% of all AD ataxias, corresponding to a prevalence of 3.1/100000 inhabitants, and 41.6/1000000 inhabitants in the Azores region. It is characterized by its onset and delay, between the ages of 35 and 40 years, and by the loss of motor functions, leading to death. Despite the monogenic cause of the disease, its pathophysiology remains unclear. A folded form of protein is a form of protein that aggregates several cellular functions, including a protein that aggregates several RNA functions. Currently, there is no cure for MJD, however, understanding the pathogenesis of the disease offers different avenues for therapeutics. This dissertation aims to review the therapeutic approach and gene therapy in MachadoJoseph Disease (MJD). Numerous studies have focused on the development of treatment studies that target a disease in different methods, preventing the action of proteins, increasing the clearance of mutant proteins and combating the mechanisms of cellular dysfunction. In recent years, it has also prevented an increasing focus on the development of gene therapies, suggesting that in the future this debilitating disease could be harnessed.

A hemoglobina é a molécula constituinte dos glóbulos vermelhos, responsável pelo transporte de oxigénio para todo o corpo. Mutações nos genes desta molécula podem resultar em alterações na sua síntese a nível quantitativo ou estrutural, afetando a sua normal função e desencadeando as hemoglobinopatias. Dentro das hemoglobinopatias existem as doenças falciformes que se particularizam pela presença obrigatória da variante hemoglobina S (Hb S). A drepanocitose, tipo de doença falciforme mais grave e incapacitante, é uma doença genética hereditária com transmissão autossómica recessiva. Caracteriza-se pela sua cronicidade, marcada por anemia e hemólise, aumentando a suscetibilidade a infeções e ao surgimento de crises vaso-oclusivas. Atualmente, é notório um crescente interesse na procura de novas terapêuticas para o tratamento deste tipo de doenças. As principais apostas a nível terapêutico recaem sobre a hidroxiureia, transfusões sanguíneas, transplante alogénico de células estaminais hematopoiéticas e a terapia genética. Porém, tendo em conta a gravidade da doença e a insuficiência relativamente às opções terapêuticas curativas, o rastreio e diagnóstico atempados constituem ferramentas valiosas para a prevenção e atuação precoce com o intuito de reduzir complicações clínicas resultantes da drepanocitose. Com a presente dissertação de mestrado pretende-se reunir, através de uma revisão bibliográfica, a mais recente evidência científica sobre a drepanocitose e as novas metodologias de diagnóstico e tratamento. A terapia genética surge como uma abordagem terapêutica promissora que já demonstrou colmatar as limitações terapêuticas anteriormente instituídas. Não obstante, terá de ser demonstrada a sua eficácia e segurança a longo prazo para se tornar no tratamento de eleição para a drepanocitose. Hemoglobin is the constituent molecule of red blood cells, responsible for transporting oxygen throughout the body. Mutations in the genes of this molecule can result in alterations in its synthesis at a quantitative or structural level, affecting its normal function and triggering hemoglobinopathies. Among the hemoglobinopathies are sickle cell diseases, which are characterized by the obligatory presence of variant hemoglobin S (Hb S). Sickle cell disease, the most serious and disabling type of sickle cell disease, is an inherited genetic disease with autosomal recessive transmission. It is characterized by its chronicity, marked by anemia and hemolysis, increasing susceptibility to infections and the onset of vaso-occlusive crises. Currently, there is a growing interest in the search for new therapies for the treatment of this type of disease. The main therapeutic options are hydroxyurea, blood transfusions, allogeneic hematopoietic stem cell transplantation and gene therapy. However, considering the severity of the disease and the insufficiency of curative therapeutic options, timely screening and diagnosis are valuable tools for prevention and early action to reduce clinical complications resulting from sickle cell disease. This master's thesis aims to gather, through a literature review, the most recent scientific evidence on sickle-cell disease and new diagnostic and treatment methodologies. Gene therapy emerges as a promising therapeutic approach that has been shown to overcome previously established therapeutic limitations. Nevertheless, its long-term efficacy and safety will have to be demonstrated to become the treatment of choice for sickle cell disease.

Doctoral Thesis (Doctoral Program Bioengineering), O cancro é uma das principais causas de morbidade e mortalidade a nível mundial, e as suas metástases são responsáveis por cerca de 90% das mortes. As atuais abordagens terapêuticas têm baixas taxas de sucesso em casos de doença metastática e a maioria dos pacientes não pode ser curada. Portanto, é crucial encontrar novas estratégias para desenvolver terapias eficientes dirigidas ao alvo. As tecnologias baseadas na seleção de aptâmeros facilitaram a descoberta de novos biomarcadores, especialmente através da tecnologia cell-SELEX, que permite a identificação de ligandos para um tipo específico de células. Deste modo, essa tecnologia foi usada para identificar um aptâmero específico para a linha celular metastática de osteossarcoma MG-63. O aptâmero mais promissor (OS-7.9) foi selecionado através de análise bioinformática, e os ensaios in vitro demonstraram a sua alta afinidade para as células-alvo, bem como para células de órgãos para os quais o osteossarcoma tem tendência a metastizar. Além disso, a tecnologia cell-SELEX foi utilizada em combinação com a sequenciação de nova geração e análise bioinformática para identificar sequências de aptâmeros (Apt1 e Apt2) com alta afinidade e especificidade para a linha celular metastática de cancro de mama (CM) MDA-MB-231. A possível translação clínica também ficou demonstrada para o Apt2 através da utilização de secções de tecido de CM. No que diz respeito aos nanossistemas de entrega de agentes terapêuticos, os sistemas não virais foram os selecionados devido à sua segurança para aplicações biomédicas. Sistemas de lipossomas catiónicos foram avaliados para o transporte de plasmídeos CRISPR/Cas9 e consequente terapia genética na linha celular usada para validação, as HEK 293T. Ensaios in vitro demonstraram elevada eficiência de transfeção e capacidade de disrupção de genes por lipoplexos à base de lípido MVL5. Estudos sobre a disrupção do microRNA-155 na linha metastática de CM Hs 578T revelaram uma ativação dos marcadores apoptóticos, bem como um aumento da paragem do ciclo celular na fase G2/M e inibição da migração. Também foram usados exossomas para a entrega de plasmídeos CRISPR/Cas9, devido à capacidade natural destas vesículas de transportar material genético. Os exossomas foram modificados com um péptido que reconhece especificamente o recetor EGFR (péptido GE11) para melhorar a internalização celular em células de CM que expressam este recetor. No geral, os resultados obtidos nesta dissertação contribuíram para o objetivo de desenvolver novas abordagens terapêuticas para cancro metastático através da identificação de novos ligandos específicos para posterior incorporação em nanossistemas de entrega, tais como lipossomas e exossomas que têm capacidade de transportar plasmídeos CRISPR/Cas9 para a aplicações de terapia genética em cancro., Cancer is a major cause of morbidity and mortality worldwide, and metastasis accounts for about 90% of cancer-related deaths. Current therapeutic approaches have poor outcomes in metastatic disease and most patients cannot be cured. Therefore, finding novel strategies is crucial for the development of new effective targeted therapies. Aptamer-based technologies have facilitated biomarker discovery, especially through cell-SELEX technology, through the identification of cell-specific ligands. Hence, this technology was used to generate an aptamer homing the highly metastatic MG-63 osteosarcoma cell line. The most promising aptamer (OS-7.9) was selected through bioinformatic analysis and in vitro assays demonstrating high affinity against the target cells, as well as against cells from common OS-metastatic sites. Moreover, the combination of cell-SELEX technology with high-throughput sequencing aided by bioinformatic analysis led to the identification of aptamer sequences (Apt1 and Apt2) with high affinity and specificity towards the metastatic breast cancer cell line MDA-MB-231. Additionally, clinical translational applicability was proven for Apt2 using breast tumor tissue sections. Regarding drug delivery systems, non-viral delivery systems were exploited due to their biosafety. Therefore, different cationic liposome formulations were evaluated for the loading of CRISPR/Cas9 plasmids and consequent gene therapy in the proof-of-concept cell line HEK 293T. In vitro assays demonstrated high transfection efficiency and gene knockout ability by MVL5-based cationic lipoplexes. Next, some preliminary data on the disruption of miR-155 in the metastatic cancer cell line Hs 578T by CRISPR/Cas9 technology showed an activation of apoptotic markers as well as an induction of G2/M cell cycle arrest, and cell migration inhibition. Ultimately, exosomes were also used as gene delivery platforms due to their natural ability to transfer genetic material. Exosomes were successfully modified with a peptide that targets EGFR receptor (GE11 peptide) to improve the cellular uptake to efficiently deliver CRISPR/Cas9 plasmids in EGFRexpressing breast cancer cells. Overall, the results gathered in this thesis contributed to the goal of developing novel therapeutic approaches for metastatic cancers through the identification of novel specific ligands for highly metastatic cancer, for posterior incorporation into gene delivery platforms such as multivalent cationic liposomes and exosomes that can carry CRISPR/Cas9 plasmids for gene cancer therapy., Acknowledge my fellowship (PD/BD/139083/2018 and COVID/BD/152644/2022) supported by the Fundação para a Ciência e Tecnologia (FCT), as well as the strategic funding of UIDB/04469/2020 unit. I would also like to thank the Centre of Biological Engineering (CEB), School of Engineering and University of Minho for providing the necessary working conditions.

Objective: A scientometric analysis produced in ophthalmic genetics and gene therapy research is lacking. The purpose of this study is to present a holistic analysis of ophthalmic genetics literature. Methods: The data used in this study were obtained from the Web of Science (WoS) Core Collection. All published documents between 1975-2019 were included. The data exported from WoS enabled the extensive details of ophthalmic genetics related literature including countries, institutions, authors, citations and keywords. Scientometric network maps of keywords and also country and institution co-authorships were created with free software. Global contributions of the countries to the ophthalmic genetics literature were shown by a graphic. Results: The search query revealed a total of 2322 documents. Most of the documents were original articles (75.75%). USA was the leading country by producing 45.39% of all documents in ophthalmic genetics research followed by UK, Germany, China and France. Pennsylvania University was the most contributing institution in the literature (5.25%) followed by University College London and Moorfields Eye Hospital. The average citations per item was 29.4. The most used keywords over a 40-year period were 'family', 'cell', 'photoreceptor' and 'expression'. Conclusions: USA and UK dominated the ophthalmic genetics research. A substantial increase in the number of published documents in this field were observed after 2010. RESUMO Objetivo: A literatura carece de análise cienciométrica produzida em genética oftálmica e de pesquisa em terapia genética. O objetivo deste estudo é apresentar uma análise holística da literatura genética oftálmica. Métodos: Os dados utilizados neste estudo foram obtidos na base de dados Web of Science (WoS) Core Collection. Todos os documentos publicados entre 1975 e 2019 foram incluídos na análise. Os dados exportados da WoS viabilizaram acesso a amplos detalhes da literatura relacionada à genética oftálmica, incluindo países, instituições, autores, citações e palavras-chave. Mapas de rede cienciométrica foram criados por meio de software gratuito, com base em palavras-chave e em coautorias de países e instituições. As contribuições globais dos países para a literatura sobre genética oftálmica foram apresentadas em gráfico. Resultados: a busca por pesquisas revelou um total de 2.322 documentos cuja maioria eram artigos originais (75,75%). Os EUA foram o país que mais produziu artigos sobre o tema, com 45,39% de todos os documentos em pesquisa genética oftálmica; ele foi seguido pelo Reino Unido, Alemanha, China e França. A Universidade da Pensilvânia foi a instituição que mais contribuiu para a literatura (5,25%), e foi seguida pela University College London e pelo Moorfields Eye Hospital. A média de citações por item foi de 29,4. As palavras-chave mais usadas em um período de 40 anos foram 'família', 'célula', 'fotorreceptor' e 'expressão'. Conclusões: Os EUA e o Reino Unido dominaram a pesquisa em genética oftálmica. Após 2010, observou-se um aumento substancial no número de documentos publicados nessa área.

The discovery of the CRISPR/Cas system and its development into a powerful genome engineering tool have revolutionized the field of molecular biology and generated excitement for its potential to treat a wide range of human diseases. As a gene therapy target, the retina offers many advantages over other tissues because of its surgical accessibility and relative immunity privilege due to its blood–retinal barrier. These features explain the large advances made in ocular gene therapy over the past decade, including the first in vivo clinical trial using CRISPR gene-editing reagents. Although viral vector-mediated therapeutic approaches have been successful, they have several shortcomings, including packaging constraints, pre-existing anti-capsid immunity and vector-induced immunogenicity, therapeutic potency and persistence, and potential genotoxicity. The use of nanomaterials in the delivery of therapeutic agents has revolutionized the way genetic materials are delivered to cells, tissues, and organs, and presents an appealing alternative to bypass the limitations of viral delivery systems. In this review, we explore the potential use of non-viral vectors as tools for gene therapy, exploring the latest advancements in nanotechnology in medicine and focusing on the nanoparticle-mediated delivery of CRIPSR genetic cargo to the retina.

Resumen El cáncer de próstata es una enfermedad prevalente, generadora de gran morbimortalidad y reportada como la quinta causa de muerte a nivel mundial. Según las estimaciones de GLOBOCAN (Global Cancer Observatory por sus siglas en inglés) para el año 2018 se reportaron 1 276 106 casos nuevos a nivel mundial. Recientemente, surgen los microARN como una posible estrategia futura como biomarcadores, tanto para el diagnóstico como para el tratamiento de la enfermedad. Los microARN son pequeñas moléculas de ARN que cumplen un papel en la regulación de la expresión génica, por lo que la expresión variable de estas moléculas tiene una función importante en la patogénesis del cáncer de próstata. La revisión de la literatura en diferentes bases de datos permitió evidenciar su papel en la patogénesis del cáncer de próstata. Se sugiere que la expresión diferencial de estas moléculas biológicas podría ser de utilidad en la práctica clínica. En Colombia se encuentra en investigación su utilidad en diferentes enfermedades, por lo cual esta revisión de tema podría contribuir a futuras investigaciones. Abstract Prostate cancer is a prevalent disease, with great morbidity and mortality, reported as the fifth leading cause of death worldwide. According to estimates for 2018 by GLOBOCAN (Global Cancer Observatory), 1 276 106 new cases of prostate cancer were reported worldwide. Identifying methods that allow an early diagnosis and treatment of the disease is necessary. MicroRNA are a possible future strategy as biomarkers for prostate cancer. These are small RNA molecules, in charge of regulating genetic expression. Their differential expression is relevant in the pathogenesis of prostate cancer. Currently, literature suggests the differential expression of these biological molecules could be a tool in prostate cancer, with clinical utility. In Colombia new research related to microRNA and prostate cancer is being conducted, which justifies the pertinence of this literature review and the contribution it can have on future research.

Today, the number of genetic diseases is around 10000 conditions, affecting to 6%-8% of all populations. This review shows us how the discovery of genetic variants in our genome, this facilitated to know with precision about the mechanisms physiopathological, and hence to recognize those target points susceptible to modifications, through therapeutical strategies different with palliative proposals, increase life expectancy, or improve qualities of life. These therapies are diverse, using drugs for polygenic diseases, nutritional therapy, special formulas, enzyme replacement therapies, hematopoietic stem cell transplant, substrate reduction, oligonucleotides, and gene therapy. These genetic diseases are heterogeneous clinically with a very low frequency; nevertheless, open to the possibility of research in new strategies for more genetic disease, that today, furthermore, are orphans. El número de enfermedades genéticas se estima que podrían ser más de 10 000 condiciones diferentes, afectando alrededor del 6-8% de la población. La presente revisión nos muestra la importancia del descubrimiento de las variantes patogénicas en nuestro genoma que nos permite conocer con mayor precisión cuales son los mecanismos fisiopatológicos, y por lo tanto conocer puntos dianas susceptibles de modificaciones, mediante diferentes estrategias terapéuticas para poder palear los síntomas y signos, aumentar la expectativa de vida, mejorando así la calidad de vida de los pacientes que tienen algunas de estas enfermedades genéticas. Las diferentes terapias que existen en la actualidad son muy diversas como fármacos de uso en patologías comunes, terapia nutricional, fórmulas especiales, terapias de reemplazo enzimático, trasplante de órganos y células hematopoyéticas, reducción de sustrato, oligonucleótidos y la terapia génica. Al ser las enfermedades genéticas clínicamente heterogéneas, abre la posibilidad de poder investigar cada vez más nuevas estrategias en un mayor número de enfermedades que en la actualidad están olvidadas. &nbsp

Il fegato è caratterizzato da un ambiente immunosoppressivo che favorisce l’attecchimento di metastasi e la proliferazione delle cellule tumorali. Trattamenti farmacologici, tra cui anche le immunoterapie, falliscono in presenza di metastasi al fegato (LMS). Ciò rende necessaria e di fondamentale importanza l’identificazione di nuovi approcci terapeutici e la caratterizzazione di bersagli chiave coinvolti nell’ambiente immunosoppressivo. I vettori lentivirali (LV), pseudotipizzati con la proteina G del virus della stomatite vescicolare e somministrati per via sistemica in topi e primati non umani, trasducono in modo efficiente le cellule del fegato, tra cui i macrofagi residenti, chiamati cellule di Kupffer (KCs). A partire da queste osservazioni, abbiamo sviluppato una nuova piattaforma basata su LV, chiamata KC-LV, per ingegnerizzare selettivamente le KC in vivo con l’obbiettivo di trasferire molecole bio-terapeutiche in modo specifico alle LMS. Per questo scopo, il disegno di KC-LV sfrutta un promotore ricostituito del recettore C di tipo 1 del mannosio (MRC1), attivo nei macrofagi, incluse le KCs. Per regolare ulteriormente la specificità per le KC, KC-LV include anche le sequenze target di microRNA che prevengono l’espressione del transgene nelle cellule endoteliali sinusoidali del fegato (LSECs) e negli epatociti. In seguito alla somministrazione sistemica di KC-LV, abbiamo osservato che l’espressione del transgene avviene in modo selettivo nelle KC, ed è particolarmente aumentata nell’area attorno alle LMS. Abbiamo poi equipaggiato KC-LV con una sequenza codificante l’IFNa, una citochina con effetti immunitari pleiotropici. L’analisi a lungo termine nei topi ha mostrato un’espressione di IFNa che dipende dalla dose di LV, prolungata e ben tollerata. Per studiarne l’efficacia terapeutica, abbiamo impiegato diversi modelli murini di LMS derivate da carcinoma del colon-retto (CRC), incluso un modello murino singenico di LMS basato su organoidi di CRC con tratti molecolari e istopatologici caratteristici della malattia umana. Il trattamento con il LV esprimente IFNa ha ritardato in modo significativo la crescita di LMS in tutti i modelli testati, raggiungendo una risposta completa nel 50% degli animali trattati. L’analisi di espressione genica a cellula singola di LMS da topi trattati con LV che esprime IFNa ha mostrato la sovra espressione dei geni che rispondono a IFN, la polarizzazione dei macrofagi verso uno stato di presentazione degli antigeni (M1-simile), associata all’espansione e a un ridotto esaurimento dei linfociti T CD8 antigene-specifici associati alle LMS. Impiegando la trascrittomica spaziale, abbiamo scoperto che l’interfaccia tra le LMS e il tessuto epatico circostante era il sito principale di azione di IFNa, con un incremento di attivazione immunitaria e aumentata presentazione dell’antigene. Confrontando le LMS di topi responsivi al trattamento con quelle di topi resistenti, abbiamo visto un accumulo di linfociti T CD8 attivati nelle lesioni responsive e invece un alto numero di cellule immunosoppressive simili a linfociti T regolatori di tipo 1 (TR1) nei topi resistenti. L’analisi molecolare suggerisce che l’infiltrazione di cellule TR1-simili è associata all’aumento di segnale di IL10 in LMS resistenti al trattamento. Riassumendo, abbiamo sviluppato un’innovativa piattaforma di terapia genica che, in seguito a una singola e ben tollerata infusione intra-vena di LV, promuove una risposta terapeutica protettiva contro le LMS attraverso l’induzione dell’attivazione immunitaria. Al contempo, abbiamo constatato che le cellule TR1-simili in questo contesto possono promuovere l’evasione immunitaria del tumore in presenza della segnalazione di IFNa, suggerendo che le cellule TR-1 simili possano divenire un potenziale nuovo obbiettivo nei casi di resistenza a immunoterapie del cancro che stimolano la segnalazione di IFNa. The liver hosts an immune suppressive environment, favoring metastatic seeding and proliferation of cancer cells. Pharmacological treatments, including immunotherapies, fail in the presence of liver metastases (LMS). Therefore, identifying new interventional tools and key targetable players involved in the immunosuppressive environment is of pivotal importance. Vesicular stomatitis virus G protein-pseudotyped lentiviral vectors (LVs) delivered systemically to mice and non-human primates efficiently transduce liver cells, including resident macrophages, termed Kupffer cells (KCs). Building on these findings, we developed a novel LV-based platform, termed KC-LV, to selectively engineer KCs in vivo with the goal of delivering bio-therapeutics specifically to LMS. To this aim, the KC-LV design exploits a reconstituted mannose receptor c type 1 (MRC1) promoter, active in macrophages, including KCs. To further fine-tune KC specificity, the KC-LV also includes microRNA target (miRT) sequences that prohibit off-target transgene expression in liver sinusoidal endothelial cells (LSECs) and hepatocytes. Upon systemic delivery of the KC-LV, we observed selective transgene expression in KCs which was enhanced in areas surrounding LMS. We then equipped the KC-LV with an IFNα-coding sequence, a cytokine with pleiotropic immune effects. Long-term analysis in mice showed LV dose-dependent, sustained and well-tolerated IFNα expression. To investigate the therapeutic efficacy, we employed different murine models of colorectal carcinoma (CRC)-derived liver metastasis (LMS) including a CRC organoid-based syngeneic mouse model of LMS containing molecular and histopathological hallmarks of the human disease. IFNα LV treatment significantly delayed LMS growth in all tested models reaching a complete response in up to 50 % of treated animals. Single cell omics of LMS from IFNα LV-treated mice showed upregulation of IFNα-responsive genes, macrophage skewing to an antigen presenting (M1-like) polarization state, and expansion as well as reduced exhaustion of LMS-associated antigen specific CD8 T cells. Employing spatial transcriptomics, we found that the interface between LMS and liver parenchymal tissue was the major site of IFNα action, which was associated with enhanced immune activation and antigen presentation. When comparing LMS of treatment responsive to resistant mice, we found accumulation of activated CD8 T-cells in responsive lesions and instead a high number of immunosuppressive T regulatory type 1 (TR1)-like cells in resistant mice. Molecular analyses suggest that TR1-like cell infiltration was associated with increased IL10 signaling in resistant LMS. In summary, we developed an innovative gene-based platform that upon a single well-tolerated intravenous LV infusion rapidly promotes a protective therapeutic response against LMS through enabling immune activation. However, we also found that TR1-like cells might promote tumor immune evasion in presence of IFNα signaling in this setting, suggesting targeting of TR1-like cells when facing resistance to cancer immunotherapies that trigger IFNα signaling.

La terapia genica (GT), il recente approccio per curare malattie genetiche rare ed ereditarie fornendo geni terapeutici in cellule mirate, ha interagito con la bioinformatica e i domini correlati all'analisi dei dati. Nelle applicazioni GT, è richiesto il monitoraggio molecolare del trattamento e sfrutta il sequenziamento di nuova generazione (NGS) per valutare l'efficacia e la sicurezza. Uno degli sforzi principali nell'identificazione dell'IS è mappare milioni di letture sequenziate nel genoma di riferimento. Oggi sono disponibili diversi allineatori per questo scopo. Tuttavia, lo scopo di alcuni lavori è ancora migliorare le prestazioni di rilevamento dell'IS tramite la scelta dell'algoritmo appropriato e testare varie strategie di filtraggio pre/post-allineamento. Poiché nonostante gli attuali miglioramenti nel campo, alcuni fattori come la lunghezza delle letture della sequenza, le regioni ripetute nei genomi di riferimento e gli errori di sequenza riducono l'accuratezza della mappatura. SI falso positivo, è un SI che viene riportato alla fine dell'identificazione SI quando non è presente. Il disallineamento è una delle possibili fonti di False Positive IS.Qui, per risolvere il problema dei False Positive e scartare il rumore nei nostri database clinici di IS, ci siamo prima concentrati sull'allineamento e sul filtraggio post-allineamento. A questo scopo, abbiamo generato una verità di base di letture simulate complessivamente composta da 125.778.685 letture di sequenziamento contenenti un numero totale di 4.333.904 IS e abbiamo provato a testare diversi passaggi della nostra pipeline bioinformatica. Abbiamo confermato che BWA-MEM, l'allineatore ampiamente utilizzato che è anche la scelta di VISPA2, ha buone prestazioni con una precisione del 91%. Ulteriori analisi hanno rivelato che alcune delle misurazioni statistiche come la qualità della mappatura (MAPQ) e il punteggio di allineamento subottimale (XS) riportate per ciascun risultato della mappatura non sono ben correlate con l'accuratezza dell'allineamento. Pertanto, i passaggi di filtraggio dopo l'allineamento potrebbero portare a scartare molte letture senza migliorare la precisione. L'eliminazione delle letture allineate correttamente potrebbe ridurre notevolmente il conteggio delle sequenze. Inoltre, sapendo che l'interazione di diversi fattori come la lunghezza di lettura, gli errori di sequenziamento, l'unicità del genoma di riferimento insieme potrebbero contribuire al risultato dell'allineamento finale, abbiamo proposto un approccio di filtraggio basato sull'albero decisionale. Il nostro risultato ha mostrato che l'algoritmo XGBoost e l'insieme di funzionalità correlate alle letture di sequenze, alle statistiche di allineamento e alle proprietà del genoma con una precisione del 95% è in grado di identificare gli artefatti delle letture sintetiche. Ciò ha suggerito che l'attuale approccio di filtraggio può essere sostituito dal nostro metodo proposto. Nella seconda parte, abbiamo mirato ad assegnare un punteggio di probabilità a ciascun IS da osservare in quella regione genomica. Per raggiungere questo obiettivo, abbiamo sfruttato le note preferenze genomiche del vettore virale e aggiunto nuove caratteristiche genomiche per generare un punteggio di probabilità. I nostri risultati su pazienti GT con due diversi background di malattia hanno mostrato AUROC > 80%. La maggior parte degli IS con punteggio di probabilità basso ha valori bassi per il conteggio delle sequenze e la ricorrenza. Ora stiamo convalidando punti di dati isolati con analisi più specifiche dell'IS. Gene Therapy (GT), the recent approach to cure rare and inherited genetic disorders by delivering therapeutic gene in targeted cells interacted with bioinformatics and data analysis-related domains. In GT applications, the molecular monitoring of the treatment is required and exploits Next generation sequencing (NGS) to assess efficacy and safety. One of the principal efforts in IS identification is mapping millions of sequenced reads to the reference genome. Today, several aligners are available for this aim. However, still improving the IS detection performance by choice of appropriate algorithm and testing various pre-/post-alignment filtering strategies is the scope of some works. Since despite the current improvements in the field, some factors like length of sequence reads, repeat regions in the reference genomes and sequence errors decrease the mapping accuracy. False Positive IS, is an IS that is reported at the end of IS identification when it is not present. Mis-alignment is one of the possible sources for False Positive IS.Here, to solve the problem of False Positive and discarding the noise in our clinical IS databases, we first focused on alignment and post-alignment filtering. For this aim, we generated a ground truth of simulated reads overall composed by 125,778,685 sequencing reads containing a total number of 4,333,904 IS and tried to test several steps of our bioinformatics pipeline. We confirmed that BWA-MEM, the widely-used aligner which is also the choice of VISPA2 has a good performance with 91% accuracy. Further analyses revealed that some of the statistic measurements such as Mapping Quality (MAPQ) and Sub optimal Alignment Score (XS) that are reported for each mapping result are not well-correlated with alignment accuracy. Therefore, filtering steps after alignment might lead to discarding many reads without improving the accuracy. Discarding reads which are aligned correctly might highly reduce sequence count. Moreover, knowing that interaction of several factors such as read length, sequencing errors, uniqueness of the reference genome together could contribute to the final alignment outcome, we proposed a decision tree-based filtering approach. Our result showed that XGBoost algorithm and the set of features which are related to sequence reads, alignment statistics and genome properties with the accuracy of 95% is able to identify artifacts of synthetic reads. This suggested that the current filtering approach can be replaced by our proposed method. In the second part, we aimed at assigning a probability score to each IS to be observed in that genomic region. To achieve this goal, we exploited the known genomic preferences of viral vector and added new genomic features to generate a probability score. Our results on GT patients with two different disease backgrounds showed AUROC > 80%. Majority of IS with low probability score have low values for sequence count and re-occurrence. We are now validating isolated data points with more IS specific analyses.

Cet article analyse la constitution d’un collectif de recherche autour d’un essai clinique de thérapie génique à la lumière du concept d’objet-frontière. Il décrit les dimensions scientifiques, organisationnelles et réglementaires de la réalisation de l’essai et interroge la nature de l’objet-frontière, entre paillasse, clinique, patients et instances réglementaires que constitue cet essai. Il analyse ensuite la façon dont le collectif réuni autour de l’essai va être mis à l’épreuve suite à la survenue d’un « événement indésirable ». Propulsées sur la scène publique, soumises à interrogation de la part d’un collectif désormais étendu, la pertinence et la solidité de l’essai deviennent l’enjeu d’une nouvelle démonstration. L’article pointe ainsi vers la nécessité de prendre en compte, dans l’analyse des collectifs de recherche et des objets-frontières qui contribuent à les fonder, les propriétés de ces objets, leur capacité à bousculer et obliger les acteurs. This paper analyzes the creation of a research collective around a gene-therapy clinical trial thanks to the concept of boundary-object. It describes the scientific, organizational and regulation dimensions of the trial, and analyzes the different boundary-objects upon which the realization of the trial is based. It then interrogates the way these boundary-objects are challenged by an unexpected event happening during the course of the trial: the pertinence and the robustness of these objects have to be demonstrated again in front of a broader audience. The argument thus points towards the necessity of taking into account the trajectory in time and public space of boundary objects in order to analyze research collectives. Este artículo analiza la constitución de un colectivo de investigación acerca de un ensayo clínico de terapia genética con la ayuda del concepto de «objeto-frontera». Describe las dimensiones científicas, organizacionales y reglamentarias de la realización del ensayo y explicita la naturaleza de los diferentes objetos-fronteras que permiten su realización. El artículo continua interrogando la manera en la cual esos objetos-fronteras están puestos a prueba con el surgimiento de un «acontecimiento indeseable» durante el ensayo. Propulsados en la escena pública, cuestionados por un colectivo desde ahora extendido, su pertinencia y su firmeza tienen que pasar por otra demonstración. El análisis subraya la necesidad de tomar en cuenta, en el análisis de los colectivos de investigación, de la trayectoria, en el tiempo y en el espacio público, de los objetos-fronteras que los constituyen.

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Advanced therapies; Clinical trials; Drug development Teràpies avançades; Assaigs clínics; Desenvolupament de fàrmacs Terapias avanzadas; Ensayos clínicos; Desarrollo de fármacos Several advanced therapy medicinal products (ATMPs) have been approved in the European Union (EU). The aim of this study is to analyse the methodological features of the clinical trials (CT) that supported the marketing authorization (MA) of the approved ATMPs in the EU. A systematic review of the characteristics of pivotal CT of ATMPs approved in the EU until January 31st, 2021 was carried out. A total of 17 ATMPs were approved and 23 CT were conducted to support the MA (median, 1, range, 1–3). Of those studies, 8 (34.78%) were non-controlled and 7 (30.43%) used historical controls. Only 7 (30.4%) were placebo or active-controlled studies. Among all CT, 21 (91.3%) were open-label and 13 (56.52%) had a single-arm design. To evaluate the primary endpoint, 18 (78.26%) studies used an intermediate and single variable. The median (IQR) number of patients enrolled in the studies was 75 (22–118). To date, ATMPs’ approval in the EU is mainly supported by uncontrolled, single-arm pivotal CT. Although there is a trend toward an adaptive or a life cycle approach, a switch to more robust clinical trial designs is expected to better define the benefit and the therapeutic added value of ATMPs.

Adenovirus; Terapia contra el cáncer; Vectores virales Adenovirus; Cancer therapy; Viral vectors Adenovirus; Teràpia contra el càncer; Vectors virals PolyPurine Reverse Hoogsteen Hairpins (PPRHs) are gene-silencing DNA-oligonucleotides developed in our laboratory that are formed by two antiparallel polypurine mirror repeat domains bound intramolecularly by Hoogsteen bonds. The aim of this work was to explore the feasibility of using viral vectors to deliver PPRHs as a gene therapy tool. After treatment with synthetic RNA, plasmid transfection, or viral infection targeting the survivin gene, viability was determined by the MTT assay, mRNA was determined by RT-qPCR, and protein levels were determined by Western blot. We showed that the RNA-PPRH induced a decrease in cell viability in a dose-dependent manner and an increase in apoptosis in PC-3 and HeLa cells. Both synthetic RNA-PPRH and RNA-PPRH intracellularly generated upon the transfection of a plasmid vector were able to reduce survivin mRNA and protein levels in PC-3 cells. An adenovirus type-5 vector encoding the PPRH against survivin was also able to decrease survivin mRNA and protein levels, leading to a reduction in HeLa cell viability. In this work, we demonstrated that PPRHs can also work as RNA species, either chemically synthesized, transcribed from a plasmid construct, or transcribed from viral vectors. Therefore, all these results are the proof of principle that viral vectors could be considered as a delivery system for PPRHs. This research was funded by grant RTI2018-093901-B-I00 from Plan Nacional de Investigación Científica (Spain). Group holding the Quality Mention from Generalitat de Catalunya 2017-SGR-94. EA is awarded with fellowships from Generalitat de Catalunya (FI).

Antipsychotics (APs) are associated with weight gain and other metabolic abnormalities such as hyperglycemia, dyslipidemia and metabolic syndrome. This translational study aimed to uncover the underlying molecular mechanisms and identify the key genes involved in AP-induced metabolic effects. An integrative gene expression analysis was performed in four different mouse tissues (striatum, liver, pancreas and adipose) after risperidone or olanzapine treatment. The analytical approach combined the identification of the gene co-expression modules related to AP treatment, gene set enrichment analysis and protein-protein interaction network construction. We found several co-expression modules of genes involved in glucose and lipid homeostasis, hormone regulation and other processes related to metabolic impairment. Among these genes, EP300, which encodes an acetyltransferase involved in transcriptional regulation, was identified as the most important hub gene overlapping the networks of both APs. Then, we explored the genetically predicted EP300 expression levels in a cohort of 226 patients with first-episode psychosis who were being treated with APs to further assess the association of this gene with metabolic alterations. The EP300 expression levels were significantly associated with increases in body weight, body mass index, total cholesterol levels, low-density lipoprotein cholesterol levels and triglyceride concentrations after 6 months of AP treatment. Taken together, our analysis identified EP300 as a key gene in AP-induced metabolic abnormalities, indicating that the dysregulation of EP300 function could be important in the development of these side effects. However, more studies are needed to disentangle the role of this gene in the mechanism of action of APs.

MYC inhibition; Targeted therapies; Epigenetics Inhibición de MYC; Terapias dirigidas; Epigenética Inhibició de MYC; Teràpies dirigides; Epigenètica The importance of MYC function in cancer was discovered in the late 1970s when the sequence of the avian retrovirus that causes myelocytic leukemia was identified. Since then, over 40 years of unceasing research have highlighted the significance of this protein in malignant transformation, especially in hematologic diseases. Indeed, some of the earliest connections among the higher expression of proto-oncogenes (such as MYC), genetic rearrangements and their relation to cancer development were made in Burkitt lymphoma, chronic myeloid leukemia and mouse plasmacytomas. Multiple myeloma (MM), in particular, is a plasma cell malignancy strictly associated with MYC deregulation, suggesting that therapeutic strategies against it would be beneficial in treating this disease. However, targeting MYC was - and, somehow, still is - challenging due to its unique properties: lack of defined three-dimensional structure, nuclear localization and absence of a targetable enzymatic pocket. Despite these difficulties, however, many studies have shown the potential therapeutic impact of direct or indirect MYC inhibition. Different molecules have been tested, in fact, in the context of MM. In this review, we summarize the current status of the different compounds, including the results of their clinical testing, and propose to continue with the efforts to identify, repurpose, redesign or improve drug candidates to combine them with standard of care therapies to overcome resistance and enable better management of myeloma treatment.

Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major technical challenge. Here, we develop a high-throughput strategy to design, screen, and optimize 5′ UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identify naturally occurring 5′ UTRs with high translation efficiencies and use this information with in silico genetic algorithms to generate synthetic 5′ UTRs. A total of ~12,000 5′ UTRs are then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identify three synthetic 5′ UTRs that outperform commonly used non-viral gene therapy plasmids in expressing protein payloads. In summary, we demonstrate that high-throughput screening of 5′ UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies., The engineering of 5′ UTRs that modulate protein expression remains a great challenge. Here we leverage synthetic biology and computational design to develop a high-throughput strategy to design, screen, and optimize 5′ UTRs that enhance protein expression for non-viral gene therapies.

A 90-day randomized feeding experiment was performed to assess the effects of dietary cobalt (Co) supplementation on the growth performance, muscle composition, status of iron and manganese in the muscle as well as the expression of growth-related genes in the muscle (myoblast determination protein 1 homolog (MyoD) and myogenin) and the stress-related gene heat shock protein 70 KDa (Hsp-70) in the liver of mahseer (Tor putitora). Feeding trial was conducted in triplicate under controlled semi-static conditions, and graded levels of dietary cobalt (0.5-3 mg/kg) were fed to six groups of advanced fry of T. putitora. The results obtained indicated a curvilinear relationship of dietary Co levels with body crude protein content and weight gain (%). A positive correlation was observed with up to 2 mg Co/kg diet. However, a decreasing trend was found with values over 2 mg Co/kg diet. The expression of muscle growth biomarkers MyoD and myogenin showed a similar response, upregulation up to 2 mg Co/kg diet and decreased expression at 3 mg Co/kg diet. Indeed, the highest dietary Co supplementation increased the expression of Hsp-70, a key gene expressed in response to stress. Moreover, the muscle content of iron and manganese showed an inverse relationship with the dietary Co supplementation. Our findings suggest that 2 mg/kg Co dietary supplementation stimulates myogenesis and optimize muscle growth and body composition, while higher levels enhanced the expression of stress response genes and impaired growth of T. putitora.