Counteracting chromatin effects of a splicing-correcting antisense oligonucleotide improves its therapeutic efficacy in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a motor-neuron disease caused by mutations of the SMN1 gene. The human paralog SMN2, whose exon 7 (E7) is predominantly skipped, cannot compensate for the lack of SMN1. Nusinersen is an antisense oligonucleotide (ASO) that upregulates E7 inclusion and SMN protein levels by displacing the splicing repressors hnRNPA1/A2 from their target site in intron 7. We show that by promoting transcriptional elongation, the histone deacetylase inhibitor VPA cooperates with a nusinersen-like ASO to promote E7 inclusion. Surprisingly, the ASO promotes the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNA polymerase II elongation that inhibits E7 inclusion. By removing the roadblock, VPA counteracts the chromatin effects of the ASO, resulting in higher E7 inclusion without large pleiotropic effects. Combined administration of the nusinersen-like ASO and VPA in SMA mice strongly synergizes SMN expression, growth, survival, and neuromuscular function.

A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2.

Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.

[Alternative splicing and the cure of spinal muscular atrophy]. / El splicing alternativo y la cura de la atrofia muscular espinal.

Alternative splicing of the messenger RNA plays a fundamental role in the flow of genetic information from DNA to proteins by expanding the coding capacity of the genome. The regulation of alternative splicing is as important as the regulation of transcription to determine the specific characteristics of cells and tissues, the normal functioning of cells and the responses of eukaryotic cells to external signals. Basic knowledge of the pre-mRNA sequences and splicing factors that recognize them has allowed scientists to design a therapeutic synthetic oligonucleotide for spinal muscular atrophy. This is an autosomal recessive inherited disease in which the SMN1 gene is mutated and affects one in 10,000 births. By blocking the binding of a negative splicing factor to the mRNA of a paralogue of the SMN1 gene, called SMN2, the Spinraza oligonucleotide corrects an abnormal alternative splicing event of the SMN2 gene and allows the synthesis of high levels of the SMN protein, constituting the first successful case of cure of a neurodegenerative disease.

El splicing alternativo del ácido ribonucleico mensajero (mRNA) juega un papel fundamental en el flujo de información genética desde el ADN a las proteínas al expandir la capacidad de codificación de los genomas. La regulación del splicing alternativo es tan importante como la regulación de la transcripción para determinar las características específicas de las células y los tejidos, el funcionamiento normal de las células y las respuestas de las células eucarióticas a las señales externas. El conocimiento básico de las secuencias del pre-mRNA y de los factores de splicing que las reconocen ha permitido a científicos diseñar un oligonucleótido sintético terapéutico para la atrofia muscular espinal. ésta es una enfermedad hereditaria autosómica recesiva en que el gen SMN1 se encuentra mutado y que afecta a uno de cada 10 000 nacimientos. Al bloquear la unión de un factor de splicing negativo al mRNA del gen parálogo del gen SMN1, denominado SMN2, el oligonucleótido Spinraza corrige un evento de splicing alternativo anormal del gen SMN2 y permite que se sinteticen altos niveles de la proteína SMN, constituyéndose en el primer caso exitoso de cura de una enfermedad neurodegenerativa.