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mRNA vaccines: Why is the biology of retroposition ignored?

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The major advantage of mRNA vaccines over more conventional approaches is their potential for rapid development and large-scale deployment in pandemic situations. In the current COVID-19 crisis the two mRNA COVID-19 vaccines have been conditionally approved and broadly applied, while others are still in clinical trials. However, there is no previous experience with the use of mRNA vaccines on the large scale in general population. This warrants a careful evaluation of mRNA vaccine safety properties by considering all available knowledge on the mRNA molecular biology and evolution. Here, I discuss the pervasive claim that mRNA-based vaccines cannot alter genomes. Surprisingly, this notion is widely stated in the mRNA vaccine literature, but never supported by referencing any primary scientific papers that would specifically address this question. This discrepancy becomes even more puzzling if one considers previous work on the molecular and evolutionary aspects of retroposition in murine and human populations that clearly documents the frequent integration of mRNA molecules into genomes, including clinical contexts. By performing basic comparisons, I showed that the sequence features of mRNA vaccines meet all known requirements for retroposition by L1 elements — the most abundant autonomously active retrotransposons in the human genome. In contrast, I found an evolutionary bias in the set of known retrocopy generating genes — a pattern that might help in the future development of retroposition-resistant therapeutic mRNAs. I conclude that is unfounded to a priori assume that mRNA-based therapeutics do not impact genomes, and that the route to genome integration of vaccine mRNAs via endogenous L1 retroelements is easily conceivable. This implies that we urgently need experimental studies that would rigorously test for the potential retroposition of vaccine mRNAs. At present, the insertional mutagenesis safety of mRNA-based vaccines should be considered unresolved.
L1-mediated retroposition. A) Retroposition cycle of L1 elements. An active L1 element is transcribed in the nucleus and resulting L1 mRNA is transported to the cytoplasm where it undergoes translation (42,43). L1 mRNA codes for ORF1 and ORF2 proteins which preferentially associate with L1 mRNA (cis-preference) to form L1 ribonucleoprotein particle (L1 RNP) (42–44). ORF1p is an RNA binding protein with chaperone activity, while ORF2p functions as reverse transcriptase and endonuclease (45,46). By a yet unresolved mechanism L1 RNP, which contains at least L1 mRNA and ORF2p, enters the nucleus. In the nucleus, L1 mRNA is reverse transcribed and integrated into the genome by the process of target-primed reverse transcription (TPRT) (43,45–47). The retroposition mechanism relies on the binding of ORF2p to the L1 mRNA poly-A tail (46,48–50). There is some evidence that the cells could uptake extracellular vesicles (EVs) containing L1 mRNA which can than undergo translation and retroposition (51). B) L1-mediated retroposition of protein coding genes. A parental protein coding gene is transcribed in the nucleus. The resulting pre-mRNA is processed and mature parental gene mRNA is then transported to the cytoplasm. L1 proteins (ORF1p and ORF2p) interact with parental gene mRNA by the process termed trans-association to form parental gene ribonucleoprotein particle (parental gene RNP) (36,43,44,47). Similar to L1 RNP, parental gene RNP enters the nucleus where by the TPRT process parental gene mRNA is reverse transcribed and integrated into the genome. The poly-A tail of parental gene mRNA plays the crucial role in this process (36,48–50). C) Hypothetical L1-mediated retroposition of vaccine mRNA. Vaccine mRNA formulated in lipid nanoparticles (LNPs) enter the cell by endocytosis (1,2,6,10,52). A fraction of vaccine mRNA enters the cytosol via endosomal escape, the rest of vaccine mRNA undergoes degradation in endosomes (52), or is repackaged in multivesicular endosomes into extracellular vesicles (EVs) and secreted back into the extracellular space (53). The neighboring or distant cells can uptake vaccine mRNA from these EVs (53,54). L1 proteins (ORF1p and ORF2p) interact with vaccine mRNA by the process termed trans-association to form vaccine mRNA ribonucleoprotein particle (vaccine mRNA RNP) (36,43,44,47). Like L1 and parental gene RNPs, vaccine mRNA RNP enters the nucleus where by the TPRT process vaccine mRNA is reverse transcribed and integrated into the genome. The poly-A tail of vaccine mRNA plays the crucial role in this process (36,48–50).
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The parental genes that generate retrocopies in human and mouse populations tend to be evolutionary ancient. The phylostratigraphic maps of human and mouse protein coding genes are generated using corresponding consensus phylogenies containing 24 internodes (phylostrata - ps). To simplify presentation of the phylostratigraphic results human and mouse phylogenies are overlapped and shown in the lower panel. The two phylogenies differ only in the last two phylostrata (ps23, ps24); i.e. Rodentia-M. musculus vs. Primates-H. sapiens lineage. Protein sequences of all human (Ensemble GRCh38.86) and mouse genes (Ensembl GRCm38.86) are compared by BLAST against the corresponding custom reference database (e-value 0.001) and mapped on the respective phylogeny using the phylostratigraphic approach (140,142,145,148). The distribution of human (483, blue numbers, (37) and mouse parental genes (1659, red numbers, (40) are shown at the top of upper panel. The log-odds chart in the upper panel shows deviation from the expected frequency of parental genes in humans (blue line) and mice (red line). Significance of these deviations is tested by the two-way hypergeometric test adjusted for multiple comparisons (*P < 0.05; **P < 0.01; ***P < 0.001). The gray shaded phylostrata (ps1 - cellular organisms, ps2 - Archaea/Asgard Archaea/Eukaryota and ps4 - Eukaryota) are enriched for parental genes. Starting with Metazoa (ps9), evolutionary more recent phylostrata show significant depletion in the number of parental genes. This phylostratigraphic pattern is effectively unchanged in the range of e-value cut-offs from 1 to 10-20, therefore it could be considered fairly robust (148).
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... These vaccines are believed to possess higher biosafety than DNA-based vaccines because the mRNA is less likely integrated into the genome than a DNA-based vaccine, as the translation of the antigens in the case of mRNA vaccines takes place in the cytoplasm and not in the nucleus, where the DNA vaccines start to work [77]. However, several studies suggest that the risk of genomic integration, even if diminished compared to DNA vaccines, also remains for those based on mRNA, considering that eukaryotic cells may exert, to some extent, a reverse transcription activity [78][79][80] that could produce DNA theoretically starting from the vaccine-delivered mRNAs [81,82]. An advantage of nucleic acid-based vaccines over protein-based vaccines is that they may lead to antigens better mimicking the viral protein structure, including the post-translational modifications. ...
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Seasonal influenza vaccines lack efficacy against drifted or pandemic influenza strains. Developing improved vaccines that elicit broader immunity remains a public health priority. Immune responses to current vaccines focus on the haemagglutinin head domain, whereas next-generation vaccines target less variable virus structures, including the haemagglutinin stem. Strategies employed to improve vaccine efficacy involve using structure-based design and nanoparticle display to optimize the antigenicity and immunogenicity of target antigens; increasing the antigen dose; using novel adjuvants; stimulating cellular immunity; and targeting other viral proteins, including neuraminidase, matrix protein 2 or nucleoprotein. Improved understanding of influenza antigen structure and immunobiology is advancing novel vaccine candidates into human trials. Current seasonal influenza vaccines lack efficacy against drifted or pandemic virus strains, and the development of novel vaccines that elicit broader immunity represents a public health priority. Here, Nabel and colleagues discuss approaches to improve vaccine efficacy which harness new insights from influenza antigen structure and human immunity, highlighting major targets, vaccines in development and ongoing challenges.
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Our genome is a historic record of successive invasions of mobile genetic elements. Like other eukaryotes, we have evolved mechanisms to limit their propagation and minimize the functional impact of new insertions. Although these mechanisms are vitally important, they are imperfect, and a handful of retroelement families remain active in modern humans. This review introduces the intrinsic functions of transposons, the tactics employed in their restraint, and the relevance of this conflict to human pathology. The most straightforward examples of disease-causing transposable elements are germline insertions that disrupt a gene and result in a monogenic disease allele. More enigmatic are the abnormal patterns of transposable element expression in disease states. Changes in transposon regulation and cellular responses to their expression have implicated these sequences in diseases as diverse as cancer, autoimmunity, and neurodegeneration. Distinguishing their epiphenomenal from their pathogenic effects may provide wholly new perspectives on our understanding of disease.
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Pseudogenes are defined as regions of the genome that contain defective copies of genes. They exist across almost all forms of life, and in mammalian genomes are annotated in similar numbers to recognized protein-coding genes. Although often presumed to lack function, growing numbers of pseudogenes are being found to play important biological roles. In consideration of their evolutionary origins and inherent limitations in genome annotation practices, we posit that pseudogenes have been classified on a scientifically unsubstantiated basis. We reflect that a broad misunderstanding of pseudogenes, perpetuated in part by the pejorative inference of the ‘pseudogene’ label, has led to their frequent dismissal from functional assessment and exclusion from genomic analyses. With the advent of technologies that simplify the study of pseudogenes, we propose that an objective reassessment of these genomic elements will reveal valuable insights into genome function and evolution. In this Perspective article, Cheetham, Faulkner and Dinger describe our latest understanding of pseudogenes, which are typically defined as defective copies of regular genes. They argue that being open minded about potential functionality, as well as carefully designing functional studies, will lead to a growing appreciation of emerging functional roles of these understudied elements.
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mRNA vaccine platforms present numerous advantages, such as versatility, rapid production, and induction of cellular and humoral responses. Moreover, mRNAs have inherent adjuvant properties due to their complex interaction with pattern recognition receptors (PRRs). This recognition can be either beneficial in activating antigen-presenting cells (APCs) or detrimental by indirectly blocking mRNA translation. To decipher this Janus effect, we describe the different innate response mechanisms triggered by mRNA molecules and how each element from the 5' cap to the poly-A tail interferes with innate/adaptive immune responses. Then, we emphasize the importance of some critical steps such as production, purification, and formulation as key events to further improve the quality of immune responses and balance innate and adaptive immunity.
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A new evaluation of previously published data suggested to us that the accumulation of mutations might slow, rather than increase, as individuals age. To explain this unexpected finding, we hypothesized that normal stem cell division rates might decrease as we age. To test this hypothesis, we evaluated cell division rates in the epithelium of human colonic, duodenal, esophageal, and posterior ethmoid sinonasal tissues. In all 4 tissues, there was a significant decrease in cell division rates with age. In contrast, cell division rates did not decrease in the colon of aged mice, and only small decreases were observed in their small intestine or esophagus. These results have important implications for understanding the relationship between normal stem cells, aging, and cancer. Moreover, they provide a plausible explanation for the enigmatic age-dependent deceleration in cancer incidence in very old humans but not in mice.
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Transposable elements are abundant in the human genome, and great strides have been made in pinpointing variations in these repetitive sequences using whole-genome sequencing. Now, the focus is shifting to understanding their expression and regulation, and the functional consequences of their insertion and retention in the genome over time. Whereas transposable element insertions have been known to cause human genetic disease since the 1980s, the scope of their contributions to heritable phenotypes is now starting to be uncovered. Here, we review the many ways human retrotransposons contribute to genome function, their dysregulation in diseases including cancer and how they affect genetic disease. Whole-genome sequencing efforts have driven our understanding of transposable elements as a source of genetic variation and the focus is now shifting to understanding their expression and regulation. This Review summarizes the possible functional consequences of transposable element insertion and retention in the genome over time, with a focus on how mobile elements cause disease.
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The Long Interspersed Nuclear Element-1 (L1) is a retrotransposon which constitutes 17% of the human genome and is associated with various diseases and aging. Estimates suggest ~100 L1 copies are capable of copying and pasting into other regions of the genome. Herein, we examined if skeletal muscle L1 markers are affected by aging or an acute bout of cycling exercise in humans. Apparently healthy younger (23±3 y/o, n=15) and older participants (58±8 y/o, n=15) donated a vastus lateralis biopsy (PRE) prior to one hour of cycling exercise at ~70% of heart rate reserve. Second (2h) and third (8h) post-exercise muscle biopsies were also obtained. L1 DNA and mRNA expression were quantified using three primer sets (5'UTR, L1.3, and ORF1). 5'UTR and L1.3 DNA methylation as well as ORF1 protein expression were also quantified. PRE 5'UTR, ORF1, or L1.3 DNA were not different between age groups (p>0.05). ORF1 mRNA was greater in older versus younger participants (p=0.014), and cycling lowered this marker at 2h versus PRE (p=0.027). 5'UTR and L1.3 DNA methylation were higher in younger versus older participants (p<0.05). Accelerometry data collected during a two-week period before the exercise bout indicated higher moderate-to-vigorous physical activity (MVPA) levels per day was associated with lower PRE ORF1 mRNA in all participants (r=-0.398, p=0.032). In summary, skeletal muscle ORF1 mRNA is higher in older apparently healthy humans which may be related to lower DNA methylation patterns. ORF1 mRNA is also reduced with endurance exercise and is negatively associated with higher daily MVPA levels.