Thioridazine reverts the phenotype in cellular and Drosophila models of amyotrophic lateral sclerosis by enhancing TDP-43 aggregate clearance.

Brain inclusions mainly composed of misfolded and aggregated TAR DNA binding protein 43 (TDP-43), are characteristic hallmarks of amyotrophic lateral sclerosis (ALS). Irrespective of the role played by the inclusions, their reduction represents an important therapeutic pathway that is worth exploring. Their removal can either lead to the recovery of TDP-43 function by removing the self-templating conformers that sequester the protein in the inclusions, and/or eliminate any potential intrinsic toxicity of the aggregates. The search for curative therapies has been hampered by the lack of ALS models for use in high-throughput screening. We adapted, optimised, and extensively characterised our previous ALS cellular model for such use. The model demonstrated efficient aggregation of endogenous TDP-43, and concomitant loss of its splicing regulation function. We provided a proof-of-principle for its eventual use in high-throughput screening using compounds of the tricyclic family and showed that recovery of TDP-43 function can be achieved by the enhanced removal of TDP-43 aggregates by these compounds. We observed that the degradation of the aggregates occurs independent of the autophagy pathway beyond autophagosome-lysosome fusion, but requires a functional proteasome pathway. The in vivo translational effect of the cellular model was tested with two of these compounds in a Drosophila model expressing a construct analogous to the cellular model, where thioridazine significantly improved the locomotive defect. Our findings have important implications as thioridazine cleared TDP-43 aggregates and recovered TDP-43 functionality. This study also highlights the importance of a two-stage, in vitro and in vivo model system to cross-check the search for small molecules that can clear TDP-43 aggregates in TDP-43 proteinopathies.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina.

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab')2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab')2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab’)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab’)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.

Innovación: consideraciones regulatorias de las vacunas de ADN / Innovation: Regulatory considerations for DNA vaccines

Las nuevas vacunas profilácticas de ADN son vacunas que se basan en la introducción de ácidos nucleicos en el organismo del huésped a inmunizar, donde dirigen la síntesis de un péptido que luego será capaz de producir una respuesta inmune protectora tanto humoral como celular. Este tipo de vacunas está diseñado para permitir una expresión localizada y breve del antígeno en cuestión y sin integración del material genético. La técnica involucra el uso de plásmidos de ADN que están conformados por un cassette de expresión codificando el/los antígenos de interés (provenientes de virus, bacterias o parásitos). Existen en la actualidad ensayos clínicos activos que evalúan la seguridad y eficacia de las vacunas de ADN tanto terapéuticas como profilácticas, siendo algunas candidatas que han avanzado hacia la fase II. La regulación de este tipo de vacunas, presenta un desafío regulatorio para las autoridades competentes en todo el mundo. Si bien existen guías para su evaluación aún se requieren estudios exhaustivos para la aprobación y uso de las mismas debido a la complejidad que presentan. La disposición vigente en Argentina contempla los requerimientos generales para este tipo de vacunas, sin embargo a futuro sería conveniente establecer nuevos lineamientos específicos para las vacunas de ADN

The new prophylactic DNA vaccines are based on the introduction of nucleic acids into the host to be immunized, in which they induce the synthesis of a peptide that can produce a protective humoral and cellular immune response. This type of vaccine is designed to allow a localized and brief expression of the antigen without genetic material integration. The technique involves the use of plasmid DNA containing an expression cassette that codifies the antigen of interest (associated to viruses, bacteria or parasites). There are many clinical assays that are evaluating safety and efficacy of prophylactic and therapeutic DNA vaccines; some of them have reached phase II trials. The regulation of this kind of vaccines represents a regulatory challenge for the competent authorities worldwide. Even though there are many guides to evaluate them, exhaustive research is still required for their approval and use due to their complexity. The current provision in Argentina considers the general requirements for this type of vaccines, however, in the future it would be advisable to establish new specific guidelines for DNA vaccines.

La agregación de TDP-43 como posible blanco terapéutico contra la Esclerosis Lateral Amiotrófica / TDP-43 aggregation as a possible therapeutic target for Amyotrophic Lateral Sclerosis / Agregação de TDP-43 como um possível alvo terapêutico para a Esclerose Lateral Amiotrófica

Los agregados de TDP-43 representan una de las característica histopatológicas más importantes de varias enfermedades neurodegenerativas, entre las que se incluye la Esclerosis Lateral Amiotrófica (ELA). TDP-43 está localizada principalmente en el núcleo. Sin embargo, los pacientes afectados por ELA presentan agregados de TDP-43 en el citoplasma de las neuronas comprometidas, con lo que se despoja al núcleo de TDP-43 funcional. Aún se desconoce si la degeneración causada por la agregación de TDP-43 es debida a una toxicidad intrínseca de los agregados o a la pérdida de función de TDP-43 como consecuencia del vaciamiento del núcleo. Varias investigaciones, incluidas las de estos autores, indican que la pérdida de función es el factor fundamental responsable de la neurodegeneración observada en presencia de inclusiones de TDP-43. Por otro lado, aún no existen tratamientos efectivos para la ELA. Por lo tanto, es de crucial importancia conocer las bases moleculares que conllevan al desarrollo de la enfermedad, con el objetivo de encontrar posibles estrategias terapéuticas. Para ello, estos autores han desarrollado un modelo celular capaz de imitar la agregación de TDP-43 y sus consecuencias. Finalmente, se ha utilizado este modelo para analizar el efecto de diferentes compuestos capaces de degradar los agregados de TDP-43 y se ha demostrado que esta podría ser una estrategia terapéutica válida para la ELA.

TDP-43 inclusions are important histopathological features of various neurodegenerative disorders, including Amyotrophic Lateral Sclerosis (ALS). TDP-43 is mainly a nuclear protein, but it shuffles from the nucleus to the cytoplasm. In patients’ brains, TDP-43 is retained in the cytoplasm of the affected motorneurons to form insoluble aggregates, which results in TDP-43 nuclear clearance. There is still no consensus whether TDP-43-mediated neurodegeneration results from a gain or loss of function of the protein or a combination of both. The work from several laboratories, including this, points towards a strong loss of function component. On the other hand, there is no effective treatment or cure for ALS. Thus, there is obviously a need to find new therapeutic strategies for ALS. In order to gain new insights into the molecular mechanism of the disease, and with the aim of looking for new methodologies that can revert it, a cellular model of TDP-43 aggregation that can mimic the phenotypic consequences found in ALS patients has been developed. Finally, this model was used to search for compounds that can dissolve these aggregates, and it was shown that the clearance of TDP-43 aggregates could be a therapeutic strategy for ALS.

Os agregados proteicos TDP-43 são características histopatológicas importantes de muitas doenças neurodegenerativas, incluindo a Esclerose Lateral Amiotrófica (ALS). A proteína TDP-43 se localiza principalmente no núcleo, porém nos cérebros de indivíduos afetados, a proteína TDP-43 fica retida no citoplasma dos neurônios motores, o que leva a formação de agregados insolúveis, resultando em deposição nuclear. Ainda não existe um consenso se a neurodegeneração mediada por TDP43 é causada por ganho ou perda da função da proteína ou uma combinação de ambos. O trabalho de muitos laboratórios, bem como este trabalho, apontam para uma forte perda da função da proteína. Por outro lado, não existe um tratamento efetivo ou cura para a ALS. Portanto, existe uma grande necessidade de identificar novos tratamentos para a ALS. Para entender o mecanismo molecular da doença, e com o objetivo de identificar novas metodologias para reverter a doença, desenvolvemos o modelo celular de agregados de TDP-43, o qual mimetiza as consequências fenotípicas encontradas em pacientes com ALS. Por fim, utilizamos esse modelo para identificar compostos que podem dissolver os agregados, e demonstramos que a liberação de inclusões de TDP-43 poderiam ser usados como tratamentos para a ALS.

A novel Drosophila model of TDP-43 proteinopathies: N-terminal sequences combined with the Q/N domain induce protein functional loss and locomotion defects.

Transactive response DNA-binding protein 43 kDa (TDP-43, also known as TBPH in Drosophila melanogaster and TARDBP in mammals) is the main protein component of the pathological inclusions observed in neurons of patients affected by different neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). The number of studies investigating the molecular mechanisms underlying neurodegeneration is constantly growing; however, the role played by TDP-43 in disease onset and progression is still unclear. A fundamental shortcoming that hampers progress is the lack of animal models showing aggregation of TDP-43 without overexpression. In this manuscript, we have extended our cellular model of aggregation to a transgenic Drosophila line. Our fly model is not based on the overexpression of a wild-type TDP-43 transgene. By contrast, we engineered a construct that includes only the specific TDP-43 amino acid sequences necessary to trigger aggregate formation and capable of trapping endogenous Drosophila TDP-43 into a non-functional insoluble form. Importantly, the resulting recombinant product lacks functional RNA recognition motifs (RRMs) and, thus, does not have specific TDP-43-physiological functions (i.e. splicing regulation ability) that might affect the animal phenotype per se. This novel Drosophila model exhibits an evident degenerative phenotype with reduced lifespan and early locomotion defects. Additionally, we show that important proteins involved in neuromuscular junction function, such as syntaxin (SYX), decrease their levels as a consequence of TDP-43 loss of function implying that the degenerative phenotype is a consequence of TDP-43 sequestration into the aggregates. Our data lend further support to the role of TDP-43 loss-of-function in the pathogenesis of neurodegenerative disorders. The novel transgenic Drosophila model presented in this study will help to gain further insight into the molecular mechanisms underlying neurodegeneration and will provide a valuable system to test potential therapeutic agents to counteract disease.

Aggregate formation prevents dTDP-43 neurotoxicity in the Drosophila melanogaster eye.

TDP-43 inclusions are an important histopathological feature in various neurodegenerative disorders, including Amyotrophic Lateral Sclerosis and Fronto-Temporal Lobar Degeneration. However, the relation of these inclusions with the pathogenesis of the disease is still unclear. In fact, the inclusions could be toxic themselves, induce loss of function by sequestering TDP-43 or a combination of both. Previously, we have developed a cellular model of aggregation using the TDP-43 Q/N rich amino acid sequence 331-369 repeated 12 times (12xQ/N) and have shown that these cellular inclusions are capable of sequestering the endogenous TDP-43 both in non-neuronal and neuronal cells. We have tested this model in vivo in the Drosophila melanogaster eye. The eye structure develops normally in the absence of dTDP-43, a fact previously seen in knock out fly strains. We show here that expression of EGFP 12xQ/N does not alter the structure of the eye. In contrast, TBPH overexpression is neurotoxic and causes necrosis and loss of function of the eye. More important, the neurotoxicity of TBPH can be abolished by its incorporation to the insoluble aggregates induced by EGFP 12xQ/N. This data indicates that aggregation is not toxic per se and instead has a protective role, modulating the functional TBPH available in the tissue. This is an important indication for the possible pathological mechanism in action on ALS patients.