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Abstract and Figures
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.
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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 . 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  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. ...
The recent development of mRNA vaccines against the SARS-CoV-2 infection has turned the spotlight on the potential of nucleic acids as innovative prophylactic agents and as diagnostic and therapeutic tools. Until now, their use has been severely limited by their reduced half-life in the biological environment and the difficulties related to their transport to target cells. These limiting aspects can now be overcome by resorting to chemical modifications in the drug and using appropriate nanocarriers, respectively. Oligonucleotides can interact with complementary sequences of nucleic acid targets, forming stable complexes and determining their loss of function. An alternative strategy uses nucleic acid aptamers that, like the antibodies, bind to specific proteins to modulate their activity. In this review, the authors will examine the recent literature on nucleic acids-based strategies in the COVID-19 era, focusing the attention on their applications for the prophylaxis of COVID-19, but also on antisense- and aptamer-based strategies directed to the diagnosis and therapy of the coronavirus pandemic.
The ongoing coronavirus disease 2019 (COVID-19) crisis has led to a new socioeconomic reality with the acquisition of novel habits. Measures imposed by governments and health authorities such as confinement and lockdown have had important consequences, including mental health problems, economic crisis, and social isolation. Combined with newly acquired habits such as hand washing, sanitization, and face masks, these have all directly and indirectly led to reduced immunity. Such effects on the immune system not only impact the epidemiological profile with respect to COVID-19 and other infectious diseases but also limit the efficacy of the ongoing anti-COVID-19 vaccination campaign. Therefore, there is a need to review these approaches and optimize measures towards better population immunity, which is much needed during such an epidemic.
Vigorous vaccination programs against SARS-CoV-2-causing Covid-19 are the major chance to fight this dreadful pandemic. The currently administered vaccines depend on adenovirus DNA vectors or on SARS-CoV-2 mRNA that might become reverse transcribed into DNA, however infrequently. In some societies, people have become sensitized against the potential short- or long-term side effects of foreign DNA being injected into humans. In my laboratory, the fate of foreign DNA in mammalian (human) cells and organisms has been investigated for many years. In this review, a summary of the results obtained will be presented. This synopsis has been put in the evolutionary context of retrotransposon insertions into pre-human genomes millions of years ago. In addition, studies on adenovirus vector-based DNA, on the fate of food-ingested DNA as well as the long-term persistence of SARS-CoV-2 RNA/DNA will be described. Actual integration of viral DNA molecules and of adenovirus vector DNA will likely be chance events whose frequency and epigenetic consequences cannot with certainty be assessed. The review also addresses problems of remaining adenoviral gene expression in adenoviral-based vectors and their role in side effects of vaccines. Eventually, it will come down to weighing the possible risks of genomic insertions of vaccine-associated foreign DNA and unknown levels of vector-carried adenoviral gene expression versus protection against the dangers of Covid-19. A decision in favor of vaccination against life-threatening disease appears prudent. Informing the public about the complexities of biology will be a reliable guide when having to reach personal decisions about vaccinations.
Mortality and morbidity from SARS-CoV2 (COVID-19) infections in children remains low, including an exceedingly low rate of horizontal and vertical transmission. However, unforeseen complications to childhood health have emerged secondary to the pandemic. Few studies to date have examined unintended complications of the pandemic in newborns and infants. In this Commentary, we discuss the impact that COVID-19 may have on inheritance of the newborn microbiome and its assembly throughout the first years of life. In the early stages of the pandemic when vertical transmission of COVID-19 was poorly understood, several studies reported increased rates of C-sections in COVID-19 positive women. Initial recommendations discouraged COVID-19 positive mothers from breastfeeding and participating in skin-to-skin care, advising them to isolate during their window of infectivity. These shifts in perinatal care can adversely impact microbial colonization during the first 1000 days of life. While obstetrical and neonatal management have evolved to reflect our current knowledge of perinatal transmission, we are observing other changes in early life exposures of infants, including increased attention to hygiene, fewer social interactions, and decreased global travel, all of which are major drivers of early-life gut colonization. Composition of the gut microbiota in adults directly impacts severity of infection, suggesting a role of microbial communities in modulating immune responses to COVID-19. Conversely, the role of the intestinal microbiome in susceptibility and severity of COVID-19 in newborns and children remains unknown. The onset of adulthood diseases is related to the establishment of a healthy gut microbiome during childhood. As we continue to define COVID-19 biology, further research is necessary to understand how acquisition of the neonatal microbiome is affected by the pandemic. Furthermore, infection control measures must be balanced with strategies that promote microbial diversity to impart optimal health outcomes and potentially modulate susceptibility of children to COVID-19.
An unresolved issue of SARS-CoV-2 disease is that patients often remain positive for viral RNA as detected by PCR many weeks after the initial infection in the absence of evidence for viral replication. We show here that SARS-CoV-2 RNA can be reverse-transcribed and integrated into the genome of the infected cell and be expressed as chimeric transcripts fusing viral with cellular sequences. Importantly, such chimeric transcripts are detected in patient-derived tissues. Our data suggest that, in some patient tissues, the majority of all viral transcripts are derived from integrated sequences. Our data provide an insight into the consequence of SARS-CoV-2 infections that may help to explain why patients can continue to produce viral RNA after recovery.
Transcript stability is associated with many biological processes, and the factors affecting mRNA stability have been extensively studied. However, little is known about the features related to human long noncoding RNA (lncRNA) stability. By inhibiting transcription and collecting samples in 10 time points, genome-wide RNA-seq studies was performed in human lung adenocarcinoma cells (A549) and RNA half-life datasets were constructed. The following observations were obtained. First, the half-life distributions of both lncRNAs and messanger RNAs (mRNAs) with one exon (lnc-human1 and m-human1) were significantly different from those of both lncRNAs and mRNAs with more than one exon (lnc-human2 and m-human2). Furthermore, some factors such as full-length transcript secondary structures played a contrary role in lnc-human1 and m-human2. Second, through the half-life comparisons of nucleus- and cytoplasm-specific and common lncRNAs and mRNAs, lncRNAs (mRNAs) in the nucleus were found to be less stable than those in the cytoplasm, which was derived from transcripts themselves rather than cellular location. Third, kmers-based protein−RNA or RNA−RNA interactions promoted lncRNA stability from lnc-human1 and decreased mRNA stability from m-human2 with high probability. Finally, through applying deep learning−based regression, a non-linear relationship was found to exist between the half-lives of lncRNAs (mRNAs) and related factors. The present study established lncRNA and mRNA half-life regulation networks in the A549 cell line and shed new light on the degradation behaviors of both lncRNAs and mRNAs.
The Coronavirus disease-19 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus -2 (SARS-CoV-2), has impacted human lives in the most profound ways with millions of infections and deaths. Scientists and pharmaceutical companies have been in race to produce vaccines against SARS-CoV-2. Vaccine generation usually demands years of developing and testing for efficacy and safety. However, it only took less than one year to generate two mRNA vaccines from their development to deployment. The rapid production time, cost-effectiveness, versatility in vaccine design, and clinically proven ability to induce cellular and humoral immune response have crowned mRNA vaccines with spotlights as most promising vaccine candidates in the fight against the pandemic. In this review, we discuss the general principles of mRNA vaccine design and working mechanisms of the vaccines, and provide an up-to-date summary of pre-clinical and clinical trials on seven anti-COVID-19 mRNA candidate vaccines, with the focus on the two mRNA vaccines already licensed for vaccination. In addition, we highlight the key strategies in designing mRNA vaccines to maximize the expression of immunogens and avoid intrinsic innate immune response. We also provide some perspective for future vaccine development against COVID-19 and other pathogens.
Sperm contributes diverse RNAs to the zygote. While sperm small RNAs have been shown to impact offspring phenotypes, our knowledge of the sperm transcriptome, especially the composition of long RNAs, has been limited by the lack of sensitive, high-throughput experimental techniques that can distinguish intact RNAs from fragmented RNAs, known to abound in sperm. Here, we integrate single-molecule long-read sequencing with short-read sequencing to detect sperm intact RNAs (spiRNAs). We identify 3440 spiRNA species in mice and 4100 in humans. The spiRNA profile consists of both mRNAs and long non-coding RNAs, is evolutionarily conserved between mice and humans, and displays an enrichment in mRNAs encoding for ribosome. In sum, we characterize the landscape of intact long RNAs in sperm, paving the way for future studies on their biogenesis and functions. Our experimental and bioinformatics approaches can be applied to other tissues and organisms to detect intact transcripts.
The phenomenon of retroposition (the reintegration of reverse-transcribed RNA into the genome) has been well studied in comparisons between species and has been identified as a source of evolutionary innovation. However, less attention has been paid to possible negative effects of retroposition. To trace the evolutionary dynamics of these negative effects, our study uses a unique genomic dataset of house mouse populations. It reveals that the initial retroposition rate is very high and that most of these newly transposed retrocopies have a deleterious impact, apparently through modifying the expression of their parental genes. In humans, this effect is expected to cause disease alleles, and we propose that genetic screening should include the search for newly transposed retrocopies.
A safe and effective vaccine against COVID-19 is urgently needed in quantities sufficient to immunise large populations. We report the preclinical development of two BNT162b vaccine candidates, which contain lipid-nanoparticle (LNP) formulated nucleoside-modified mRNA encoding SARS-CoV-2 spike glycoprotein-derived immunogens. BNT162b1 encodes a soluble, secreted, trimerised receptor-binding domain (RBD-foldon). BNT162b2 encodes the full-length transmembrane spike glycoprotein, locked in its prefusion conformation (P2 S). The flexibly tethered RBDs of the RBD-foldon bind ACE2 with high avidity. Approximately 20% of the P 2S trimers are in the two-RBD ‘down,’ one-RBD ‘up’ state. In mice, one intramuscular dose of either candidate elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong TH1 CD4+ and IFNγ+ CD8+ T-cell responses. Prime/boost vaccination of rhesus macaques with BNT162b candidates elicits SARS-CoV-2 neutralising geometric mean titres 8.2 to 18.2 times that of a SARS-CoV-2 convalescent human serum panel. The vaccine candidates protect macaques from SARS-CoV-2 challenge, with BNT162b2 protecting the lower respiratory tract from the presence of viral RNA and with no evidence of disease enhancement. Both candidates are being evaluated in phase 1 trials in Germany and the United States1–3. BNT162b2 is being evaluated in an ongoing global, pivotal Phase 2/3 trial (NCT04380701, NCT04368728).
The global research and development of mRNA vaccines have been prodigious over the past decade, and the work in this field has been stimulated by the urgent need for rapid development of vaccines in response to an emergent disease such as the current COVID-19 pandemic. Nevertheless, there remain gaps in our understanding of the mechanism of action of mRNA vaccines, as well as their long-term performance in areas such as safety and efficacy. This paper reviews the technologies and processes used for developing mRNA prophylactic vaccines, the current status of vaccine development, and discusses the immune responses induced by mRNA vaccines. It also discusses important issues with regard to the evaluation of mRNA vaccines from regulatory perspectives. Setting global norms and standards for biologicals including vaccines to assure their quality, safety and efficacy has been a WHO mandate and a core function for more than 70 years. New initiatives are ongoing at WHO to arrive at a broad consensus to formulate international guidance on the manufacture and quality control, as well as nonclinical and clinical evaluation of mRNA vaccines, which is deemed necessary to facilitate international convergence of manufacturing and regulatory practices and provide support to National Regulatory Authorities in WHO member states.
In this concise review, we summarize the concepts behind mRNA vaccination. We discuss the innate and adaptive immune response generated by mRNA vaccines in different animal models and in humans. We give examples of viral infections where mRNA vaccines have shown to induce potent responses and we discuss in more detail the recent SARS-CoV-2 mRNA vaccine trials in humans.
The COVID-19 pandemic has the potential to affect the human microbiome in infected and uninfected individuals, having a substantial impact on human health over the long term. This pandemic intersects with a decades-long decline in microbial diversity and ancestral microbes due to hygiene, antibiotics, and urban living (the hygiene hypothesis). High-risk groups succumbing to COVID-19 include those with preexisting conditions, such as diabetes and obesity, which are also associated with microbiome abnormalities. Current pandemic control measures and practices will have broad, uneven, and potentially long-term effects for the human microbiome across the planet, given the implementation of physical separation, extensive hygiene, travel barriers, and other measures that influence overall microbial loss and inability for reinoculation. Although much remains uncertain or unknown about the virus and its consequences, implementing pandemic control practices could significantly affect the microbiome. In this Perspective, we explore many facets of COVID-19−induced societal changes and their possible effects on the microbiome, and discuss current and future challenges regarding the interplay between this pandemic and the microbiome. Recent recognition of the microbiome’s influence on human health makes it critical to consider both how the microbiome, shaped by biosocial processes, affects susceptibility to the coronavirus and, conversely, how COVID-19 disease and prevention measures may affect the microbiome. This knowledge may prove key in prevention and treatment, and long-term biological and social outcomes of this pandemic.
We integrated ubiquity, mass and lifespan of all major cell types to achieve a comprehensive quantitative description of cellular turnover. We found a total cellular mass turnover of 80 ± 20 grams per day, dominated by blood cells and gut epithelial cells. In terms of cell numbers, close to 90% of the (0.33 ± 0.02) × 1012 cells per day turnover was blood cells. A comprehensive census of the dynamics of death and regeneration of cells and tissues provides an estimation of the distribution of cellular turnover in the human body.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the resulting coronavirus disease 2019 (Covid-19) have afflicted tens of millions of people in a worldwide pandemic. Safe and effective vaccines are needed urgently.
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In an ongoing multinational, placebo-controlled, observer-blinded, pivotal efficacy trial, we randomly assigned persons 16 years of age or older in a 1:1 ratio to receive two doses, 21 days apart, of either placebo or the BNT162b2 vaccine candidate (30 μg per dose). BNT162b2 is a lipid nanoparticle–formulated, nucleoside-modified RNA vaccine that encodes a prefusion stabilized, membrane-anchored SARS-CoV-2 full-length spike protein. The primary end points were efficacy of the vaccine against laboratory-confirmed Covid-19 and safety.
A total of 43,548 participants underwent randomization, of whom 43,448 received injections: 21,720 with BNT162b2 and 21,728 with placebo. There were 8 cases of Covid-19 with onset at least 7 days after the second dose among participants assigned to receive BNT162b2 and 162 cases among those assigned to placebo; BNT162b2 was 95% effective in preventing Covid-19 (95% credible interval, 90.3 to 97.6). Similar vaccine efficacy (generally 90 to 100%) was observed across subgroups defined by age, sex, race, ethnicity, baseline body-mass index, and the presence of coexisting conditions. Among 10 cases of severe Covid-19 with onset after the first dose, 9 occurred in placebo recipients and 1 in a BNT162b2 recipient. The safety profile of BNT162b2 was characterized by short-term, mild-to-moderate pain at the injection site, fatigue, and headache. The incidence of serious adverse events was low and was similar in the vaccine and placebo groups.
A two-dose regimen of BNT162b2 conferred 95% protection against Covid-19 in persons 16 years of age or older. Safety over a median of 2 months was similar to that of other viral vaccines. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04368728.)
Messenger RNA (mRNA) represents a promising next-generation approach for both treatment and vaccination. Lipid based particles are one of the most investigated delivery systems for mRNA formulations. Here we explore how the complexing lipid affects uptake and translation of lipoplex-delivered RNA in resident cells in human skin explants and, we explore a more modular delivery system that utilizes mRNA added to pre-formed nanoparticles prior to dosing. We prepared formulations of lipoplexes with ionizable, cationic or zwitterionic lipids, externally complexed these with mRNA, and observed which cells internalized and/or expressed the mRNA over 72 h after intradermal injections into primary, human, skin explants. Using a flow cytometry panel to assess cellular phenotypes, mRNA uptake and mRNA expression, we found that, unlike other cell types, adipocytes expressed mRNA efficiently at 4 and 24 h after mRNA-lipoplex injection and contributed the greatest proportion of total RNA-encoded protein expression, despite being the lowest frequency cell type. Other cell types (epithelial cells, fibroblasts, T cells, B cells, dendritic cells, monocytes, NK cells, Langerhans cells, and leukocytes) had increasing mRNA expression over the course of 72 h, irrespective of lipoplex formulation. We observed that overall charge of the particle, but not the complexing lipid classification, was predictive for the pattern of mRNA uptake and expression among resident cell types in this model.
This study provides insight into maximizing protein expression, using modular mRNA lipoplexes that are more compatible with product development, in a clinically relevant, human skin explant model.
There are concepts in science that need time to overcome initial disbelief before finally arriving at the moment when they are embraced by the research community. One of these concepts is the biological meaning of the small, spheroidal vesicles released from cells, which are described in the literature as microparticles, microvesicles, or exosomes. In the beginning, this research was difficult, as it was hard to distinguish these small vesicles from cell debris or apoptotic bodies. However, they may represent the first language of cell-cell communication, which existed before a more specific intercellular cross-talk between ligands and receptors emerged during evolution. In this review article, we will use the term "extracellular microvesicles" (ExMVs) to refer to these small spheroidal blebs of different sizes surrounded by a lipid layer of membrane. We have accepted an invitation from the Editor-in-Chief to write this review in observance of the 20th anniversary of the 2001 ASH Meeting when our team demonstrated that, by horizontal transfer of several bioactive molecules, including mRNA species and proteins, ExMVs harvested from embryonic stem cells could modify hematopoietic stem/progenitor cells and expand them ex vivo. Interestingly, the result that moved ExMV research forward was published first in 2005 in Leukemia, having been previously rejected by other major scientific journals out of simple disbelief. Therefore, the best judge of a new concept is the passage of time, although the speed of its adoption is aided by perseverance and confidence in one's own data. In this perspective article, we will provide a brief update on the current status of, hopes for, and likely future of ExMV research as well as therapeutic and diagnostic applications, with a special emphasis on hematopoiesis.
Correspondence between evolution and development has been discussed for more than two centuries. Recent work reveals that phylogeny-ontogeny correlations are indeed present in developmental transcriptomes of eukaryotic clades with complex multicellularity. Nevertheless, it has been largely ignored that the pervasive presence of phylogeny-ontogeny correlations is a hallmark of development in eukaryotes. This perspective opens a possibility to look for similar parallelisms in biological settings where developmental logic and multicellular complexity are more obscure. For instance, it has been increasingly recognized that multicellular behaviour underlies biofilm formation in bacteria. However, it remains unclear whether bacterial biofilm growth shares some basic principles with development in complex eukaryotes. Here we show that the ontogeny of growing Bacillus subtilis biofilms recapitulates phylogeny at the expression level. Using time-resolved transcriptome and proteome profiles, we found that biofilm ontogeny correlates with the evolutionary measures, in a way that evolutionary younger and more diverged genes were increasingly expressed towards later timepoints of biofilm growth. Molecular and morphological signatures also revealed that biofilm growth is highly regulated and organized into discrete ontogenetic stages, analogous to those of eukaryotic embryos. Together, this suggests that biofilm formation in Bacillus is a bona fide developmental process comparable to organismal development in animals, plants and fungi. Given that most cells on Earth reside in the form of biofilms and that biofilms represent the oldest known fossils, we anticipate that the widely-adopted vision of the first life as a single-cell and free-living organism needs rethinking.
A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the global coronavirus infectious disease (COVID-19) public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved the expression and immunogenicity of betacoronavirus spike proteins1. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here we show that mRNA-1273 induces both potent neutralizing antibody responses to wild-type (D614) and D614G mutant2 SARS-CoV-2 and CD8 T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in Phase 3 efficacy evaluation.
Synthetic messenger RNA (mRNA) tools often use pseudouridine and 5-methyl cytidine as substitutions for uridine and cytidine to avoid the immune response and cytotoxicity induced by introducing mRNA into cells. However, the influence of base modifications on the functionality of the RNA tools is poorly understood. Here we show that synthetic mRNA switches containing N1-methylpseudouridine (m1Ψ) as a substitution of uridine substantially out-performed all other modified bases studied, exhibiting enhanced microRNA and protein sensitivity, better cell-type separation ability, and comparably low immune stimulation. We found that the observed phenomena stem from the high protein expression from m1Ψ containing mRNA and efficient translational repression in the presence of target microRNAs or proteins. In addition, synthetic gene circuits with m1Ψ significantly improve performance in cells. These findings indicate that synthetic mRNAs with m1Ψ modification have enormous potentials in the research and application of biofunctional RNA tools.
Bacilli can form dormant, highly resistant, and metabolically inactive spores to cope with extreme environmental challenges. In this study, we examined the evolutionary age of Bacillus subtilis sporulation genes using the approach known as genomic phylostratigraphy. We found that B. subtilis sporulation genes cluster in several groups that emerged at distant evolutionary time-points, suggesting that the sporulation process underwent several stages of expansion. Next, we asked whether such evolutionary stratification of the genome could be used to predict involvement in sporulation of presently uncharacterized genes (y-genes). We individually inactivated a representative sample of uncharacterized genes that arose during the same evolutionary periods as the known sporulation genes and tested the resulting strains for sporulation phenotypes. Sporulation was significantly affected in 16 out of 37 (43%) tested strains. In addition to expanding the knowledge base on B. subtilis sporulation, our findings suggest that evolutionary age could be used to help with genome mining.
Transposable elements (TEs) are sequences currently or historically mobile, and are present across all eukaryotic genomes. A growing interest in understanding the regulation and function of TEs has revealed seemingly dichotomous roles for these elements in evolution, development, and disease. On the one hand, many gene regulatory networks owe their organization to the spread of cis‐elements and DNA binding sites through TE mobilization during evolution. On the other hand, the uncontrolled activity of transposons can generate mutations and contribute to disease, including cancer, while their increased expression may also trigger immune pathways that result in inflammation or senescence. Interestingly, TEs have recently been found to have novel essential functions during mammalian development. Here, the function and regulation of TEs are discussed, with a focus on LINE1 in mammals. It is proposed that LINE1 is a beneficial endogenous dual regulator of gene expression and genomic diversity during mammalian development, and that both of these functions may be detrimental if deregulated in disease contexts. The recent results pointing to a dual role of transposable elements (TEs) as both mutagens and regulators of mammalian development are discussed. Several cellular pathways keep the mutagenic activity of TEs under control, but these pathways can be disrupted by stress. It is proposed that TEs are versatile regulators of chromatin biology that can contribute to evolvability of development.
About half of all cancers have somatic integrations of retrotransposons. Here, to characterize their role in oncogenesis, we analyzed the patterns and mechanisms of somatic retrotransposition in 2,954 cancer genomes from 38 histological cancer subtypes within the framework of the Pan-Cancer Analysis of Whole Genomes (PCAWG) project. We identified 19,166 somatically acquired retrotransposition events, which affected 35% of samples and spanned a range of event types. Long interspersed nuclear element (LINE-1; L1 hereafter) insertions emerged as the first most frequent type of somatic structural variation in esophageal adenocarcinoma, and the second most frequent in head-and-neck and colorectal cancers. Aberrant L1 integrations can delete megabase-scale regions of a chromosome, which sometimes leads to the removal of tumor-suppressor genes, and can induce complex translocations and large-scale duplications. Somatic retrotranspositions can also initiate breakage–fusion–bridge cycles, leading to high-level amplification of oncogenes. These observations illuminate a relevant role of L1 retrotransposition in remodeling the cancer genome, with potential implications for the development of human tumors. An analysis of 2,954 genomes from 38 cancer subtypes identified 19,166 retrotransposition events in 35% of samples. Aberrant LINE-1 retrotranspositions can lead to the deletion of tumor-suppressor genes as well as the amplification of oncogenes.
mRNA technologies have the potential to transform areas of medicine, including the prophylaxis of infectious diseases. The advantages for vaccines range from the acceleration of immunogen discovery to rapid response and multiple disease target manufacturing. A greater understanding of quality attributes that dictate translation efficiency, as well as a comprehensive appreciation of the importance of mRNA delivery, are influencing a new era of investment in development activities. The application of translational sciences and growing early-phase clinical experience continue to inform candidate vaccine selection. Here we review the state of the art for the prevention of infectious diseases by using mRNA and pertinent topics to the biotechnology and pharmaceutical industries.
In the past few years, there has been increasing focus on the use of messenger RNA (mRNA) as a new therapeutic modality. Current clinical efforts encompassing mRNA-based drugs are directed toward infectious disease vaccines, cancer immunotherapies, therapeutic protein replacement therapies, and treatment of genetic diseases. However, challenges that impede the successful translation of these molecules into drugs are that (i) mRNA is a very large molecule, (ii) it is intrinsically unstable and prone to degradation by nucleases, and (iii) it activates the immune system. Although some of these challenges have been partially solved by means of chemical modification of the mRNA, intracellular delivery of mRNA still represents a major hurdle. The clinical translation of mRNA-based therapeutics requires delivery technologies that can ensure stabilization of mRNA under physiological conditions. Here, we (i) review opportunities and challenges in the delivery of mRNA-based therapeutics with a focus on non-viral delivery systems, (ii) present the clinical status of mRNA vaccines, and (iii) highlight perspectives on the future of this promising new type of medicine.
Virus–host cell interactions initiate a host cell–defensive response during virus infection. How transposable elements in the host cell respond to viral stress at the molecular level remains largely unclear. By reanalyzing next generation sequencing data sets from dozens of virus infection studies from the Gene Expression Omnibus database, we found that genome-wide transposon expression up-regulation in host cells occurs near antiviral response genes and exists in all studies regardless of virus, species, and host cell tissue types. Some transposons were found to be up-regulated almost immediately upon infection and before increases in virus replication and significant increases in interferon β expression. These findings indicate that transposon up-regulation is a common phenomenon during virus infection in human and mouse and that early up-regulated transposons are part of the first wave response during virus infection.
Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used 2 approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs, to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly expressed mRNAs contained a highly structured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide variety of primary sequences. Using a set of eGFP mRNAs with independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e., mRNA being actively translated). This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability.
Mobile genetic Elements (MEs) are segments of DNA which can copy themselves and other transcribed sequences through the process of retrotransposition (RT). In humans several disorders have been attributed to RT, but the role of RT in severe developmental disorders (DD) has not yet been explored. Here we identify RT-derived events in 9738 exome sequenced trios with DD-affected probands. We ascertain 9 de novo MEs, 4 of which are likely causative of the patient’s symptoms (0.04%), as well as 2 de novo gene retroduplications. Beyond identifying likely diagnostic RT events, we estimate genome-wide germline ME mutation rate and selective constraint and demonstrate that coding RT events have signatures of purifying selection equivalent to those of truncating mutations. Overall, our analysis represents a comprehensive interrogation of the impact of retrotransposition on protein coding genes and a framework for future evolutionary and disease studies.
Long interspersed element-1s (L1s) encode 2 proteins (ORF1p and ORF2p) that preferentially mobilize (i.e., retrotranspose) their encoding messenger RNA (mRNA) transcript. ORF1p and/or ORF2p can also mobilize other cellular RNAs, including short interspersed elements (SINEs), U6 small nuclear RNA (snRNA), and mRNAs. Here, we demonstrate the RNA ligase RtcB can join U6 snRNA to L1 or other cellular RNAs to create chimeric RNAs; retrotransposition of the resultant chimeric RNAs leads to chimeric pseudogene formation; and chimeric U6/L1 RNAs are part of the transcriptome in multiple human cells. These data suggest RNA ligation contributes to the plasticity of the transcriptome and that the retrotransposition of chimeric RNAs can generate genetic variation in the human genome.
The active uptake of exogenous nucleic acids by spermatozoa of virtually all animal species is a well-established phenomenon whose significance has long been underappreciated. A growing body of published data demonstrates that extracellular vesicles released from mammalian somatic tissues pass an RNA-based flow of information to epididymal spermatozoa, thereby crossing the Weismann barrier. That information is delivered to oocytes at fertilization and affects the fate of the developing progeny. We propose that this essential process of epigenetic transmission depends upon the documented ability of epididymal spermatozoa to bind and internalize foreign nucleic acids in their nuclei. In other words, spermatozoa are not passive vectors of exogenous molecules but rather active participants in essential somatic communication across generations.
In the early nineties, pioneering steps were taken in the use of mRNA as a therapeutic tool for vaccination. In the following decades, an improved understanding of the mRNA pharmacology, together with novel insights in immunology have positioned mRNA-based technologies as next-generation vaccines. This review outlines the history and current state-of-the-art in mRNA vaccination, while presenting an immunological view on mRNA vaccine development. As such, we highlight the challenges in vaccine design, testing and administration, key considerations in the design of mRNA-based vaccines and new opportunities that arise when packaging mRNA in nanoparticulate vaccines. Finally, we discuss the mRNA self-adjuvant effect as a critical, but dichotomous parameter that determines the safety, efficacy and strength of the evoked immune response.
Although messenger RNAs are key molecules for understanding life, until now, no method has existed to determine the full-length sequence of endogenous mRNAs including their poly(A) tails. Moreover, although non-A nucleotides can be incorporated in poly(A) tails, there also exists no method to accurately sequence them. Here, we present full-length poly(A) and mRNA sequencing (FLAM-seq), a rapid and simple method for high-quality sequencing of entire mRNAs. We report a complementary DNA library preparation method coupled to single-molecule sequencing to perform FLAM-seq. Using human cell lines, brain organoids and Caenorhabditis elegans we show that FLAM-seq delivers high-quality full-length mRNA sequences for thousands of different genes per sample. We find that 3′ untranslated region length is correlated with poly(A) tail length, that alternative polyadenylation sites and alternative promoters for the same gene are linked to different tail lengths, and that tails contain a substantial number of cytosines.
Long interspersed element-1 (LINE-1 or L1) retrotransposons replicate through a copy-and-paste mechanism using an RNA intermediate. However, little is known about the physical transmission of retrotransposon RNA between cells. To examine the horizontal transfer of an active human L1 retrotransposon mediated by extracellular vesicles (EVs), human cancer cells were transfected with an expression construct containing a retrotransposition-competent human L1 tagged with a reporter gene. Using this model, active retrotransposition events were detected by screening for the expression of the reporter gene inserted into the host genome by retrotransposition. EVs including exosomes and microvesicles were isolated from cells by differential centrifugation. The enrichment of L1-derived reporter RNA transcripts were detected in EVs isolated from cells expressing active L1 retrotransposition. The delivery of reporter RNA was confirmed in recipient cells, and reporter genes were detected in the genome of recipient cells. Additionally, employing qRT-PCR, we found that host-encoded factors are activated in response to increased exposure to L1-derived RNA transcripts in recipient cells. Our results suggest that the horizontal transfer of retrotransposons can occur through the incorporation of RNA intermediates delivered via EVs and may have important implications for the intercellular regulation of gene expression and gene function.
Different pathogenic germline mutations in the RET oncogene are identified in MEN 2, a hereditary syndrome characterized by medullary thyroid carcinoma (MTC) and other endocrine tumors. Although genetic predisposition is recognized, not all RET mutation carriers will develop the disease during their lifetime or, likewise, RET mutation carriers belonging to the same family may present clinical heterogeneity. It has been suggested that a single germline mutation might not be sufficient for development of MEN 2-associated tumors and a somatic bi-allelic alteration might be required. Here we investigated the presence of somatic second hit mutation in the RET gene in MTC.
We integrated Multiplex Ligation-dependent Probe Amplification (MLPA) and whole exome sequencing (WES) to search for copy number alteration (CNA) in the RET gene in MTC samples and medullary thyroid cell lines (TT and MZ-CR-1). We next found reads spanning exon-exon boundaries on RET, an indicative of retrocopy. We subsequently searched for RET retrocopies in the human reference genome (GRCh37) and in the 1000 Genomes Project data, by looking for reads reporting joined exons in the RET locus or distinct genomic regions. To determine RET retrocopy specificity and recurrence, DNA isolated from sporadic and MEN 2-associated MTC (n = 37), peripheral blood (n = 3) and papillary thyroid carcinomas with RET fusion (n = 10) samples were tested using PCR-sequencing methodology.
Through MLPA we have found evidence of CNA in the RET gene in MTC samples and MTC cell lines. WES analysis reinforced the presence of the CNA and hinted for a retroposed copy of RET not found in the human reference genome and 1.000 Genomes Project. Extended analysis confirmed the presence of a somatic MTC-related retrocopy of RET in both sporadic and hereditary tumors. We further unveiled a recurrent (28%) novel point mutation (p.G548 V) found exclusively in the retrocopy of RET. The mutation was also found in cDNA of mutated samples, suggesting it might be functional.
We here report a somatic specific RET retroposed copy in MTC samples and cell lines. Our results support the idea that generation of retrocopies in somatic cells is likely to contribute to MTC genesis and progression.
In less than one year since the outbreak of the COVID-19 pandemic, two mRNA-based vaccines, BNT162b2 and mRNA-1273, were granted the first historic authorization for emergency use, while another mRNA vaccine, CVnCoV, progressed to phase 3 clinical testing. The COVID-19 mRNA vaccines represent a new class of vaccine products, which consist of synthetic mRNA strands encoding the SARS-CoV-2 Spike glycoprotein, packaged in lipid nanoparticles to deliver mRNA to cells. This review digs deeper into the scientific breakthroughs of the last decades that laid the foundations for the rapid rise of mRNA vaccines during the COVID-19 pandemic. As well as providing momentum for mRNA vaccines, SARS-CoV-2 represents an ideal case study allowing to compare design-activity differences between the different mRNA vaccine candidates. Therefore, a detailed overview of the composition and (pre)clinical performance of the three most advanced mRNA vaccines is provided and the influence of choices in their structural design on to their immunogenicity and reactogenicity profile is discussed in depth. In addition to the new fundamental insights in the mRNA vaccines' mode of action highlighted here, we also point out which unknowns remain that require further investigation and possibly, optimization in future mRNA vaccine development.
The devastating SARS-CoV-2 pandemic demands rapid vaccine development and large scale production to meet worldwide needs. mRNA vaccines have emerged as one of the most promising technologies to address this unprecedented challenge. Here, we show preclinical data for our clinical candidate CVnCoV, a lipid nanoparticle (LNP) encapsulated non-modified mRNA vaccine that encodes the full length, pre-fusion stabilised SARS-CoV-2 Spike (S) protein. S translated from CVnCoV is cleaved, post-translationally modified, and presented on the cell surface, highlighting the ability of mRNA vaccines to mimic antigen presentation during viral infection. Immunisation with CVnCoV induced strong humoral responses with high titres of virus neutralizing antibodies in mice and hamsters and robust CD4 ⁺ and CD8 ⁺ T cell responses in mice. Most importantly, vaccination with CVnCoV fully protected hamster lungs from challenge with wild type SARS-CoV-2. To gain insights in the risk of vaccine-enhanced disease, hamsters vaccinated with a suboptimal dose of CVnCoV leading to breakthrough viral replication were analysed for signs of vaccine-enhanced disease. No evidence of increased viral replication or exacerbated inflammation and damage to viral target organs was detectable, giving strong evidence for a favourable safety profile of CVnCoV. Overall, data presented here provide evidence that CVnCoV represents a potent and safe vaccine candidate against SARS-CoV-2.
The first proof-of-concept studies about the feasibility of genetic vaccines were published over three decades ago, opening the way for future development. The idea of nonviral antigen delivery had multiple advantages over the traditional live or inactivated pathogen-based vaccines, but a great deal of effort had to be invested to turn the idea of genetic vaccination into reality. Although early proof-of-concept studies were groundbreaking, they also showed that numerous aspects of genetic vaccines needed to be improved. Until the early 2000s, the vast majority of effort was invested into the development of DNA vaccines due to the potential issues of instability and low in vivo translatability of messenger RNA (mRNA). In recent years, numerous studies have demonstrated the outstanding abilities of mRNA to elicit potent immune responses against infectious pathogens and different types of cancer, making it a viable platform for vaccine development. Multiple mRNA vaccine platforms have been developed and evaluated in small and large animals and humans and the results seem to be promising. RNA-based vaccines have important advantages over other vaccine approaches including outstanding efficacy, safety, and the potential for rapid, inexpensive, and scalable production. There is a substantial investment by new mRNA companies into the development of mRNA therapeutics, particularly vaccines, increasing the number of basic and translational research publications and human clinical trials underway. This review gives a broad overview about genetic vaccines and mainly focuses on the past and present of mRNA vaccines along with the future directions to bring this potent vaccine platform closer to therapeutic use.
There has been an urgent need of vaccines against coronavirus disease 2019 (COVID-19) due to the ongoing SARS-CoV-2 pandemic. Among all approaches, messenger RNA (mRNA) -based vaccine has emerged as a rapid and versatile platform to quickly respond to such a challenge. Here, we developed a lipid-nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (termed ARCoV). Intramuscular immunization of ARCoV mRNA-LNPs elicited robust neutralizing antibodies against SARS-CoV-2 as well as Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse adapted strain. Additionally, ARCoV was manufactured in liquid formulation and can be stored at room temperature for at least one week. This novel COVID-19 mRNA vaccine, ARCoV, is currently being evaluated in phase 1 clinical trials.
It is now clear that cells form a wide collection of large RNA-protein assemblies, referred to as RNP granules. RNP granules exist in bacterial cells and can be found in both the cytosol and nucleus of eukaryotic cells. Recent approaches have begun to define the RNA and protein composition of a number of RNP granules. Herein, we review the composition and assembly of RNP granules, as well as how RNPs are targeted to RNP granules using stress granules and P-bodies as model systems. Taken together, these reveal that RNP granules form through the summative effects of a combination of protein-protein, protein-RNA, and RNA-RNA interactions. Similarly, the partitioning of individual RNPs into stress granules is determined by the combinatorial effects of multiple elements. Thus, RNP granules are assemblies generally dominated by combinatorial effects, thereby providing rich opportunities for biological regulation.
Messenger RNA (mRNA) vaccines represent a relatively new vaccine class showing great promise for the future. This optimism is built on recently published studies demonstrating the efficacy of mRNA vaccines in combatting several types of cancer and infectious pathogens where conventional vaccine platforms may fail to induce protective immune responses. These results would not have been possible without critical recent innovations in the field, such as the development of safe and efficient materials for in vivo mRNA delivery and advanced protocols for the production of high quality mRNA. This review summarizes the most important developments in mRNA vaccines from the past few years and discusses the challenges and future directions for the field.
Long INterspersed element (LINE‐1, L1) retrotransposons are the most abundant transposable elements in the human genome, constituting approximately 17%. They move by a “copy‐paste” mechanism, involving reverse transcription of an RNA intermediate and insertion of its cDNA copy at a new site in the genome. L1 retrotransposition (L1‐RTP) can cause insertional mutations, alter gene expression, transduce exons, and induce epigenetic dysregulation. L1‐RTP is generally repressed, however a number of observations collected over about fifteen years revealed that it can occur in response to environmental stresses. Moreover, emerging evidence indicates that L1‐RTP can play a role in the onset of several neurological and oncological diseases in humans. In recent years, great attention has been paid to the exposome paradigm, which proposes that health effects of an environmental factor should be evaluated considering both cumulative environmental exposures and the endogenous processes resulting from the biological response. L1‐RTP could be an endogenous process considered for this application. Here, we summarize the current understanding of environmental factors that can affect the retrotransposition of human L1 elements. Evidence indicates that L1‐RTP alteration is triggered by numerous and various environmental stressors, such as chemical agents (heavy metals, carcinogens, oxidants, drugs), physical agents (ionizing and non‐ionizing radiations) and experiential factors (voluntary exercise, social isolation, maternal care, environmental light/dark cycles). These data come from in vitro studies on cell lines, and in vivo studies on transgenic animals: future investigations should be focused on physiologically relevant models to gain a better understanding of this topic. This article is protected by copyright. All rights reserved.
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.
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.
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.
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.
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.
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.
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.