RESUMO
Therapeutic applications of synthetic mRNA were proposed more than 30 years ago, and are currently the basis of one of the vaccine platforms used at a massive scale as part of the public health strategy to get COVID-19 under control. To date, there are no published studies on the biodistribution, cellular uptake, endosomal escape, translation rates, functional half-life and inactivation kinetics of synthetic mRNA, rates and duration of vaccine-induced antigen expression in different cell types. Furthermore, despite the assumption that there is no possibility of genomic integration of therapeutic synthetic mRNA, only one recent study has examined interactions between vaccine mRNA and the genome of transfected cells, and reported that an endogenous retrotransposon, LINE-1 is unsilenced following mRNA entry to the cell, leading to reverse transcription of full length vaccine mRNA sequences, and nuclear entry. This finding should be a major safety concern, given the possibility of synthetic mRNA-driven epigenetic and genomic modifications arising. We propose that in susceptible individuals, cytosolic clearance of nucleotide modified synthetic (nms-mRNAs) is impeded. Sustained presence of nms-mRNA in the cytoplasm deregulates and activates endogenous transposable elements (TEs), causing some of the mRNA copies to be reverse transcribed. The cytosolic accumulation of the nms-mRNA and the reverse transcribed cDNA molecules activates RNA and DNA sensory pathways. Their concurrent activation initiates a synchronized innate response against non-self nucleic acids, prompting type-I interferon and pro-inflammatory cytokine production which, if unregulated, leads to autoinflammatory and autoimmune conditions, while activated TEs increase the risk of insertional mutagenesis of the reverse transcribed molecules, which can disrupt coding regions, enhance the risk of mutations in tumour suppressor genes, and lead to sustained DNA damage. Susceptible individuals would then expectedly have an increased risk of DNA damage, chronic autoinflammation, autoimmunity and cancer. In light of the current mass administration of nms-mRNA vaccines, it is essential and urgent to fully understand the intracellular cascades initiated by cellular uptake of synthetic mRNA and the consequences of these molecular events.
RESUMO
Production of asparaginase II of Saccharomyces cerevisiae is regulated by nitrogen and can be used as a model system for studying other secreted proteins in yeast. Green fluorescent protein (GFP) from Aequorea victoria was fused to the carboxy-terminus of the enzyme by genomic integration to the locus ASP3 of S. cerevisiae. We determined asparaginase II activity, mRNA ASP3, mRNA ASP3-GFP and GFP fluorescence. Nitrogen starvation in cells carrying the chimera ASP3-GFP caused an increase in fluorescence and in the expression of ASP3. We have shown that cells producing the chimera Asp3-GFPp displayed the same response to nitrogen starvation as control cells. We demonstrated that Asp3-GFPp can be used for studying asparaginase II secretion under nitrogen starvation in vivo.
A produção de asparaginase II de Saccharomyces cerevisiae é regulada por nitrogênio e pode ser utilizada como um sistema modelo para estudar outras proteínas secretadas, em leveduras. A proteína "green fluorescent protein" (GFP) de Aequorea victoria foi fusionada à porção carboxi-terminal de Asp3p por integração genômica da sequência de GFP ao locus ASP3. Determinaram-se os níveis de atividade de asparaginase II, mRNA ASP3, mRNA ASP3-GFP e de fluorescência para GFP. A depleção para nitrogênio, em células portadoras do gene quimérico ASP3-GFP, fez aumentar a fluorescência, assim como a expressão de ASP3. Demonstramos que Asp3-GFPp pode ser utilizada para estudar a secreção de asparaginase II em células submetidas à privação de nitrogênio in vivo.