Modified mRNA-Mediated CCN5 Gene Transfer Ameliorates Cardiac Dysfunction and Fibrosis without Adverse Structural Remodeling.

Modified mRNAs (modRNAs) are an emerging delivery method for gene therapy. The success of modRNA-based COVID-19 vaccines has demonstrated that modRNA is a safe and effective therapeutic tool. Moreover, modRNA has the potential to treat various human diseases, including cardiac dysfunction. Acute myocardial infarction (MI) is a major cardiac disorder that currently lacks curative treatment options, and MI is commonly accompanied by fibrosis and impaired cardiac function. Our group previously demonstrated that the matricellular protein CCN5 inhibits cardiac fibrosis (CF) and mitigates cardiac dysfunction. However, it remains unclear whether early intervention of CF under stress conditions is beneficial or more detrimental due to potential adverse effects such as left ventricular (LV) rupture. We hypothesized that CCN5 would alleviate the adverse effects of myocardial infarction (MI) through its anti-fibrotic properties under stress conditions. To induce the rapid expression of CCN5, ModRNA-CCN5 was synthesized and administrated directly into the myocardium in a mouse MI model. To evaluate CCN5 activity, we established two independent experimental schemes: (1) preventive intervention and (2) therapeutic intervention. Functional analyses, including echocardiography and magnetic resonance imaging (MRI), along with molecular assays, demonstrated that modRNA-mediated CCN5 gene transfer significantly attenuated cardiac fibrosis and improved cardiac function in both preventive and therapeutic models, without causing left ventricular rupture or any adverse cardiac remodeling. In conclusion, early intervention in CF by ModRNA-CCN5 gene transfer is an efficient and safe therapeutic modality for treating MI-induced heart failure.

Modified mRNA Therapeutics for Heart Diseases.

Cardiovascular diseases (CVD) remain a substantial global health problem and the leading cause of death worldwide. Although many conventional small-molecule treatments are available to support the cardiac function of the patient with CVD, they are not effective as a cure. Among potential targets for gene therapy are severe cardiac and peripheral ischemia, heart failure, vein graft failure, and some forms of dyslipidemias. In the last three decades, multiple gene therapy tools have been used for heart diseases caused by proteins, plasmids, adenovirus, and adeno-associated viruses (AAV), but these remain as unmet clinical needs. These gene therapy methods are ineffective due to poor and uncontrolled gene expression, low stability, immunogenicity, and transfection efficiency. The synthetic modified mRNA (modRNA) presents a novel gene therapy approach which provides a transient, stable, safe, non-immunogenic, controlled mRNA delivery to the heart tissue without any risk of genomic integration, and achieves a therapeutic effect in different organs, including the heart. The mRNA translation starts in minutes, and remains stable for 8-10 days (pulse-like kinetics). The pulse-like expression of modRNA in the heart induces cardiac repair, cardiomyocyte proliferation and survival, and inhibits cardiomyocyte apoptosis post-myocardial infarction (MI). Cell-specific (cardiomyocyte) modRNA translation developments established cell-specific modRNA therapeutics for heart diseases. With these laudable characteristics, combined with its expression kinetics in the heart, modRNA has become an attractive therapeutic for the treatment of CVD. This review discusses new developments in modRNA therapy for heart diseases.

The creation of an endovascular exit through the vessel wall using a minimally invasive working channel in order to reach all human organs.

Since the establishment of the Seldinger technique for secure entry to the vascular system, there has been a rapid evolution in imaging and catheters that has made the arteries and veins internal routes to any place in the body for interventions. It is curious that a general exit from the vasculature in a similar manner has not been proposed earlier. Possibly, the simplest reason is that accidental perforation of the vasculature by guide wire or catheter is a feared adverse event in endovascular intervention. Most places in the body can be reached by ultrasonography or computed tomography-guided intervention. Some organs such as the central nervous system, the heart and pancreas are harder to access and, in some organs, like the kidney, repeated percutaneous punctions to cover large areas is not suitable. We present a new general purpose micro-endovascular device creating a working channel to these 'hard to reach' organs by an inverted Seldinger technique. This review details this trans-vessel wall technique, which has been studied in pancreas for transplantation of insulin-producing cells, for injection of contrast agent to the heart and to the brain, bowels and kidney in rat, rabbit, swine and macaque monkeys with up to one year of follow-up without adverse events. Furthermore, the payloads that can be given through such a system are briefly discussed. Drugs, cells, gene vectors and other therapeutic substances may be injected directly to the tissue to increase efficacy and decrease risk of off-site adverse effects.

Potential Application of Modified mRNA in Cardiac Regeneration.

Heart failure remains the leading cause of human death worldwide. After a heart attack, the formation of scar tissue due to the massive death of cardiomyocytes leads to heart failure and sudden death in most cases. In addition, the regenerative ability of the adult heart is limited after injury, partly due to cell-cycle arrest in cardiomyocytes. In the current post-COVID-19 era, urgently authorized modified mRNA (modRNA) vaccines have been widely used to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Therefore, modRNA-based protein replacement may act as an alternative strategy for improving heart disease. It is a safe, effective, transient, low-immunogenic, and integration-free strategy for in vivo protein expression, in addition to recombinant protein and stem-cell regenerative therapies. In this review, we provide a summary of various cardiac factors that have been utilized with the modRNA method to enhance cardiovascular regeneration, cardiomyocyte proliferation, fibrosis inhibition, and apoptosis inhibition. We further discuss other cardiac factors, modRNA delivery methods, and injection methods using the modRNA approach to explore their application potential in heart disease. Factors for promoting cardiomyocyte proliferation such as a cocktail of three genes comprising FoxM1, Id1, and Jnk3-shRNA (FIJs), gp130, and melatonin have potential to be applied in the modRNA approach. We also discuss the current challenges with respect to modRNA-based cardiac regenerative medicine that need to be overcome to apply this approach to heart disease. This review provides a short description for investigators interested in the development of alternative cardiac regenerative medicines using the modRNA platform.

Cell-Specific mRNA Therapeutics for Cardiovascular Diseases and Regeneration.

Cardiovascular diseases (CVDs) represent a significant global health burden, demanding innovative therapeutic approaches. In recent years, mRNA therapeutics have emerged as a promising strategy to combat CVDs effectively. Unlike conventional small-molecule drugs, mRNA therapeutics enable the direct modulation of cellular functions by delivering specific mRNA molecules to target cells. This approach offers unprecedented advantages, including the ability to harness endogenous cellular machinery for protein synthesis, thus allowing precise control over gene expression without insertion into the genome. This review summarizes the current status of the potential of cell-specific mRNA therapeutics in the context of cardiovascular diseases. First, it outlines the challenges associated with traditional CVD treatments and emphasizes the need for targeted therapies. Subsequently, it elucidates the underlying principles of mRNA therapeutics and the development of advanced delivery systems to ensure cell-specificity and enhanced efficacy. Notably, innovative delivery methods such as lipid nanoparticles and exosomes have shown promise in improving the targeted delivery of mRNA to cardiac cells, activated fibroblasts, and other relevant cell types. Furthermore, the review highlights the diverse applications of cell-specific mRNA therapeutics in addressing various aspects of cardiovascular diseases, including atherosclerosis, myocardial infarction, heart failure, and arrhythmias. By modulating key regulatory genes involved in cardiomyocyte proliferation, inflammation, angiogenesis, tissue repair, and cell survival, mRNA therapeutics hold the potential to intervene at multiple stages of CVD pathogenesis. Despite its immense potential, this abstract acknowledges the challenges in translating cell-specific mRNA therapeutics from preclinical studies to clinical applications like off-target effects and delivery. In conclusion, cell-specific mRNA therapeutics have emerged as a revolutionary gene therapy approach for CVD, offering targeted interventions with the potential to significantly improve patient outcomes.

A Real-World Nationwide Study on COVID-19 Trend in Italy during the Autumn-Winter Season of 2020 (before Mass Vaccination) and 2021 (after Mass Vaccination) Integrated with a Retrospective Analysis of the Mortality Burden per Year.

SARS-CoV-2 virulence is known to increase with lowering of environmental temperature and solar ultraviolet radiation; therefore, we have focused our real-world nationwide study concerning with COVID-19 trend and dynamics on the coldest seasons of the year in Italy, the Western country hardest hit at the onset of the pandemic, comparing the autumn-winter of 2020 (before mass vaccination but when the emergency machinery was fully operative in terms of tracing and swabs) with the autumn-winter of 2021 (after mass vaccination), and analyzing the mortality burden by age groups and life stages in the years 2019 (pre-COVID-19), 2020 (before mass vaccination), and 2021 (after mass vaccination). METHODS: During the state of national health emergency, the Civil Defense Department released the aggregate data coming from the Higher Institute of Health, the Ministry of Health, the Italian Regions, and the Independent Provinces, to inform the population about the pandemic situation, daily. Among these data, there were the number of contagions, performed swabs, hospitalizations in Intensive Care Units (ICU), non-ICU patients, and deaths. By means of a team effort, we have collected and elaborated all these data, comparing the COVID-19 pandemic in Italy during the autumn-winter of 2020 with the autumn-winter of 2021. Moreover, we have extracted from the database of the National Institute of Statistics the total number of annual deaths in Italy during the years 2019, 2020, and 2021, comparing them to each other in order to evaluate the mortality burden attributable to COVID-19. RESULTS: From the autumn-winter of 2020 to the autumn-winter of 2021, the contagions increased by ≈285%, against a ≈290% increase in the performed swabs; therefore, the mean positivity rate passed from 8.74% before mass vaccination to 8.59% after mass vaccination. The unprecedent vaccination campaign allowed a ≈251% abatement in COVID-19 deaths, and a reduction of ≈224% and ≈228% in daily ICU and non-ICU hospitalizations due to COVID-19, respectively. Regarding COVID-19 deaths, in 2020, there was a mortality excess of ≈14.3% quantifiable in 105,900 more deaths compared to 2019, the pre-COVID-19 year; 103,183 out of 105,900 deaths occurred in older adults (≥60 years), which is equivalent to ≈97.4%, while in adults over 50, the segment of population just below older adults, in 2020, there were 2807 more deaths than in 2019. Surprisingly, from the analysis of our data, it is emerged that in people under the age of 40 in the years 2019, 2020, and 2021, there were 7103, 6808, and 7165 deaths, respectively. This means that in subjects under 40 during 2020, there were 295 fewer deaths than in 2019, while during 2021, there were 357 more deaths than in 2020, equivalent to ≈5.2% more. CONCLUSIONS: COVID-19 is a potential life-threatening disease mainly in older adults, as they are the most vulnerable due to inherent immunosenescence and inflammaging. Extensive vaccination in this segment of population with up-to-date vaccines is the means to reduce deaths, hospitalizations, and ICU pressure in the public interest. In the event of future threats, a new mass vaccination campaign should not be implemented without taking into account the individual age; it should primarily be aimed at people over 60 and at patients of any age with immune deficits, and secondly at people over 50. COVID-19 vaccination shows a favorable benefit-risk ratio in older adults, while the balance steps down under the age of 40; this younger segment of the population should be therefore exempt from any mandatory vaccination.

Endovascular transplantation of mRNA-enhanced mesenchymal stromal cells results in superior therapeutic protein expression in swine heart.

Heart failure has a poor prognosis and no curative treatment exists. Clinical trials are investigating gene- and cell-based therapies to improve cardiac function. The safe and efficient delivery of these therapies to solid organs is challenging. Herein, we demonstrate the feasibility of using an endovascular intramyocardial delivery approach to safely administer mRNA drug products and perform cell transplantation procedures in swine. Using a trans-vessel wall (TW) device, we delivered chemically modified mRNAs (modRNA) and mRNA-enhanced mesenchymal stromal cells expressing vascular endothelial growth factor A (VEGF-A) directly to the heart. We monitored and mapped the cellular distribution, protein expression, and safety tolerability of such an approach. The delivery of modRNA-enhanced cells via the TW device with different flow rates and cell concentrations marginally affect cell viability and protein expression in situ. Implanted cells were found within the myocardium for at least 3 days following administration, without the use of immunomodulation and minimal impact on tissue integrity. Finally, we could increase the protein expression of VEGF-A over 500-fold in the heart using a cell-mediated modRNA delivery system compared with modRNA delivered in saline solution. Ultimately, this method paves the way for future research to pioneer new treatments for cardiac disease.

Optimization of Strategy for Modified mRNA Inducing Cardiac-Specific Expression in Mice.

Lipid nanoparticle (LNP)-coated-modified RNA(modRNA) has been developed for enhancing the stability of modRNA, but it tends to accumulate in liver. The current study aimed to optimize strategy for increasing cardiac expression efficiency of modRNA. We synthesized Luciferase (Luc)-modRNA, and also developed 122Luc modRNA, a liver silencing Luc modRNA. Intramyocardial injection of naked Luc modRNA induced high bioluminescence signal in heart, but very low in other organs including liver. Luc modRNA-LNP injection showed the signal was increased by 5 folds in the heart and by 15,000 folds in the liver, compared to naked Luc modRNA group. In comparison with Luc modRNA-LNP group, the liver signal was decreased to 0.17%, while cardiac signal showed a slight drop by intramyocardial injection of 122Luc-modRNA-LNP. Our data revealed that intramyocardial injection of naked modRNA could effectively induced cardiac-specific expression. For cardiac delivery of Luc modRNA-LNP, 122modRNA-LNP enhances specificity of cardiac expression by abolishing liver signal.

Safe and Effective Delivery of mRNA Using Modified PEI-Based Lipopolymers.

Chemically modified mRNA (modRNA) has proven to be a versatile tool for the treatment of various cancers and infectious diseases due to recent technological advancements. However, a safe and effective delivery system to overcome the complex extracellular and intracellular barriers is required in order to achieve higher therapeutic efficacy and broaden clinical applications. Here, we explored All-Fect and Leu-Fect C as novel transfection reagents derived from lipopolymers, which demonstrated excellent biocompatibility, efficient delivery capabilities, and a robust ability to escape the lysosomes. These properties directly increase mRNA stability by preventing mRNA degradation by nucleases and simultaneously promote efficient gene translation in vitro and in vivo. The modRNA delivered with lipopolymer vectors sustained effective transfection in mouse hearts following direct intramyocardial injection, as well as in major organs (liver and spleen) after systemic administration. No observable immune reactions or systemic toxicity were detected following the systemic administration of lipopolymer-mRNA complexes to additional solid organs. This study identified commercial reagents for the effective delivery of modRNA and may help facilitate the advancement of gene-based interventions involving the safe and effective delivery of nucleic acid drug substances.

Engineered exosomes enriched in netrin-1 modRNA promote axonal growth in spinal cord injury by attenuating inflammation and pyroptosis.

BACKGROUND: Spinal cord injury (SCI) brings a heavy burden to individuals and society, and there is no effective treatment at present. Exosomes (EX) are cell secreted vesicles containing molecules such as nucleic acids and proteins, which hold promise for the treatment of SCI. Netrin-1 is an axon guidance factor that regulates neuronal growth. We investigated the effects of engineered EX enriched in netrin-1 chemically synthetic modified message RNA (modRNA) in treating SCI in an attempt to find a novel therapeutic approach for SCI. METHODS: Netrin-1 modRNA was transfected into bone marrow mesenchymal stem cells to obtain EX enriched with netrin-1 (EX-netrin1). We built an inflammatory model in vitro with lipopolysaccharide (LPS) in vitro to study the therapeutic effect of EX-netrin1 on SCI. For experiments in vitro, ELISA, CCK-8 assay, immunofluorescence staining, lactate dehydrogenase release experiments test, real-time quantitative polymerase chain reaction, and western blot were conducted. At the same time, we constructed a rat model of SCI. MRI, hematoxylin-eosin and Nissl staining were used to assess the extent of SCI in rats. RESULTS: In vitro experiments showed that EX had no effect on the viability of oligodendrocytes and PC12 cells. EX-netrin1 could attenuate LPS-induced inflammation and pyroptosis and accelerate axonal/dentritic growth in PC12 cells/oligodendrocytes. In addition, netrin-1 could activate the PI3K/AKT/mTOR signalling pathway upon binding to its receptor unc5b. When Unc5b and PI3K were inhibited, the effect of EX-netrin1 was weakened, which could be reversed by PI3K or mTOR activator. Our in vivo experiments indicated that EX-netrin1 could promote recovery in rats with SCI. CONCLUSION: We found that EX-netrin1 regulated inflammation, pyroptosis and axon growth in SCI via the Unc5b/PI3K/AKT/mTOR pathway, which provides a new strategy for the treatment of SCI.

mRNA in the Context of Protein Replacement Therapy.

Protein replacement therapy is an umbrella term used for medical treatments that aim to substitute or replenish specific protein deficiencies that result either from the protein being absent or non-functional due to mutations in affected patients. Traditionally, such an approach requires a well characterized but arduous and expensive protein production procedure that employs in vitro expression and translation of the pharmaceutical protein in host cells, followed by extensive purification steps. In the wake of the SARS-CoV-2 pandemic, mRNA-based pharmaceuticals were recruited to achieve rapid in vivo production of antigens, proving that the in vivo translation of exogenously administered mRNA is nowadays a viable therapeutic option. In addition, the urgency of the situation and worldwide demand for mRNA-based medicine has led to an evolution in relevant technologies, such as in vitro transcription and nanolipid carriers. In this review, we present preclinical and clinical applications of mRNA as a tool for protein replacement therapy, alongside with information pertaining to the manufacture of modified mRNA through in vitro transcription, carriers employed for its intracellular delivery and critical quality attributes pertaining to the finished product.

IL-10 modified mRNA monotherapy prolongs survival after composite facial allografting through the induction of mixed chimerism.

Vascularized composite allotransplantation has great potential in face transplantation by supporting functional restoration following tissue grafting. However, the need for lifelong administration of immunosuppressive drugs still limits its wide use. Modified mRNA (modRNA) technology provides an efficient and safe method to directly produce protein in vivo. Nevertheless, the use of IL-10 modRNA-based protein replacement, which exhibits anti-inflammatory properties, has not been shown to prolong composite facial allograft survival. In this study, IL-10 modRNA was demonstrated to produce functional IL-10 protein in vitro, which inhibited pro-inflammatory cytokines and in vivo formation of an anti-inflammatory environments. We found that without any immunosuppression, C57BL/6J mice with fully major histocompatibility complex (MHC)-mismatched facial allografts and local injection of IL-10 modRNA had a significantly prolonged survival rate. Decreased lymphocyte infiltration and pro-inflammatory T helper 1 subsets and increased anti-inflammatory regulatory T cells (Tregs) were seen in IL-10 modRNA-treated mice. Moreover, IL-10 modRNA induced multilineage chimerism, especially the development of donor Treg chimerism, which protected allografts from destruction because of recipient alloimmunity. These results support the use of monotherapy based on immunomodulatory IL-10 cytokines encoded by modRNA, which inhibit acute rejection and prolong allograft survival through the induction of donor Treg chimerism.

Engineered adipose-derived stem cells with IGF-1-modified mRNA ameliorates osteoarthritis development.

BACKGROUND: Osteoarthritis (OA), a prevalent degenerative disease characterized by degradation of extracellular matrix (ECM), still lacks effective disease-modifying therapy. Mesenchymal stem cells (MSCs) transplantation has been regarded as the most promising approach for OA treatment while engrafting cells alone might not be adequate for effective regeneration. Genetic modification has been used to optimize MSC-based therapy; however, there are still significant limitations that prevent the clinical translation of this therapy including low efficacy and safety concerns. Recently, chemically modified mRNA (modRNA) represents a promising alternative for the gene-enhanced MSC therapy. In this regard, we hypothesized that adipose derived stem cells (ADSCs) engineered with modRNA encoding insulin-like growth factor 1 (IGF-1) were superior to native ADSCs on ameliorating OA development. METHODS: Mouse ADSCs were acquired from adipose tissue and transfected with modRNAs. First, the kinetics and efficacy of modRNA-mediated gene transfer in mouse ADSCs were analyzed in vitro. Next, we applied an indirect co-culture system to analyze the pro-anabolic potential of IGF-1 modRNA engineered ADSCs (named as IGF-1-ADSCs) on chondrocytes. Finally, we evaluated the cell retention and chondroprotective effect of IGF-1-ADSCs in vivo using fluorescent labeling, histology and immunohistochemistry. RESULTS: modRNA transfected mouse ADSCs with high efficiency (85 ± 5%) and the IGF-1 modRNA-transfected ADSCs facilitated burst-like production of bio-functional IGF-1 protein. In vitro, IGF-1-ADSCs induced increased anabolic markers expression of chondrocytes in inflammation environment compared to untreated ADSCs. In a murine OA model, histological and immunohistochemical analysis of knee joints harvested at 4 weeks and 8 weeks after OA induction suggested IGF-1-ADSCs had superior therapeutic effect over native ADSCs demonstrated by lower histological OARSI score and decreased loss of cartilage ECM. CONCLUSIONS: These findings collectively supported the therapeutic potential of IGF-1-ADSCs for clinical OA management and cartilage repair.

Modified mRNA-Based Vaccines Against Coronavirus Disease 2019.

The pandemic of coronavirus disease 2019 (COVID-19) continuously causes deaths worldwide, representing a considerable challenge to health care and economic systems with a new precedent in human history. Many therapeutic medicines primarily focused on preventing severe organ damage and complications, which can be fatal in some confirmed cases. The synthesized modified mRNA (modRNA) represents a nonviral, integration-free, zero-footprint, efficient, and safe strategy for vaccine discovery. modRNA-based technology has facilitated the rapid development of the first COVID-19 vaccines due to its cost- and time-saving properties, thus initiating a new era of prophylactic vaccines against infectious diseases. Recently, COVID-19 modRNA vaccines were approved, and a large-scale vaccination campaign began worldwide. To date, results suggest that the modRNA vaccines are highly effective against virus infection, which causes COVID-19. Although short-term studies have reported that their safety is acceptable, long-term safety and protective immunity remain unclear. In this review, we describe two major approved modRNA vaccines and discuss their potential myocarditis complications.

Cubital Tunnel Syndrome Temporally after COVID-19 Vaccination.

Coronavirus disease 2019 (COVID-19) is the most dramatic pandemic of the new millennium. To counter it, specific vaccines have been launched in record time under emergency use authorization or conditional marketing authorization and have been subjected to additional monitoring. The European Medicines Agency recommend reporting any suspected adverse reactions during this additional monitoring phase. For the first time in the available medical literature, we report a left cubital tunnel syndrome in a 28-year-old right-handed healthy male after seven days from the first dose of Spikevax® (formerly Moderna COVID-19 Vaccine). Histochemistry for Alcian Blue performed on the tissue harvested from the cubital site reveals myxoid degeneration of the small nerve collaterals, a clear sign of nerve injury. It still remains unclear why the syndrome occurs in a localized and not generalized form to all osteofibrous tunnels. Today, modified messenger ribonucleic acid vaccines as Spikevax® represent an avantgarde technological platform with a lot of potential, but one which needs careful monitoring in order to identify in advance those patients who may experience adverse events after their administration.

Robust genome editing via modRNA-based Cas9 or base editor in human pluripotent stem cells.

CRISPR systems have revolutionized biomedical research because they offer an unprecedented opportunity for genome editing. However, a bottleneck of applying CRISPR systems in human pluripotent stem cells (hPSCs) is how to deliver CRISPR effectors easily and efficiently. Here, we developed modified mRNA (modRNA)-based CRIPSR systems that utilized Cas9 and p53DD or a base editor (ABE8e) modRNA for the purposes of knocking out genes in hPSCs via simple lipid-based transfection. ABE8e modRNA was employed to disrupt the splice donor site, resulting in defective splicing of the target transcript and ultimately leading to gene knockout. Using our modRNA CRISPR systems, we achieved 73.3% ± 11.2% and 69.6 ± 3.8% knockout efficiency with Cas9 plus p53DD modRNA and ABE8e modRNA, respectively, which was significantly higher than the plasmid-based systems. In summary, we demonstrate that our non-integrating modRNA-based CRISPR methods hold great promise as more efficient and accessible techniques for genome editing of hPSCs.

A Three-Case Series of Thrombotic Deaths in Patients over 50 with Comorbidities Temporally after modRNA COVID-19 Vaccination.

Coronavirus disease 2019 (COVID-19) is the most dramatic pandemic of the new millennium; to counteract it, specific vaccines have been launched in record time under emergency use authorization or conditional marketing authorization by virtue of a favorable risk/benefit balance. Among the various technological platforms, there is that exploiting a nucleoside-modified messenger RNA (modRNA), such as Comirnaty®, and that which is adenoviral vector-based. In the ongoing pharmacovigilance, the product information of the latter has been updated about the risk of thrombotic thrombocytopenia, venous thromboembolism without thrombocytopenia and immune thrombocytopenia without thrombosis. However, from an in-depth literature review, the same adverse events can rarely occur with modRNA vaccines too. In support of this, we here report a three-case series of thrombotic deaths in patients over 50 with comorbidities temporally after Comirnaty®, investigated by means of post-mortem histopathology and immunohistochemistry. In two out of three cases, the cause of death is traced back to pulmonary microthromboses rich in activated platelets, quite similar morphologically to those described in patients who died from severe COVID-19. Even if remote in the face of millions of administered doses, clinicians should be aware of the possible thrombotic risk also after Comirnaty®, in order to avoid a misdiagnosis with potentially lethal consequences. Since COVID-19 vaccines are inoculated in subjects to be protected, maximum attention must be paid to their safety, and prophylactic measures to increase it are always welcome. In light of the evidence, the product information of modRNA COVID-19 vaccines should be updated about the thrombotic risk, as happened for adenoviral vector-based vaccines.

Apropos of menstrual changes and abnormal uterine bleeding after COVID-19 vaccination.

It is news of 28 October 2022 that the Pharmacovigilance Risk Assessment Committee of the European Medicines Agency has recommended to add heavy menstrual bleeding among the side effects of unknown frequency inside the package insert of nucleoside-modified messenger ribonucleic acid vaccines to prevent coronavirus disease 2019 (COVID-19). The decision has been made in the light of the numerous reports of unexpected menstrual changes or abnormal uterine bleeding following COVID-19 vaccination. Here we advance a possible involvement of the particular adenohypophyseal microcirculation in these strange and still unexplained events.

Lipid Nanoparticles for Organ-Specific mRNA Therapeutic Delivery.

Advances in the using in vitro transcribed (IVT) modRNA in the past two decades, especially the tremendous recent success of mRNA vaccines against SARS-CoV-2, have brought increased attention to IVT mRNA technology. Despite its well-known use in infectious disease vaccines, IVT modRNA technology is being investigated mainly in cancer immunotherapy and protein replacement therapy, with ongoing clinical trials in both areas. One of the main barriers to progressing mRNA therapeutics to the clinic is determining how to deliver mRNA to target cells and protect it from degradation. Over the years, many different vehicles have been developed to tackle this issue. Desirable vehicles must be safe, stable and preferably organ specific for successful mRNA delivery to clinically relevant cells and tissues. In this review we discuss various mRNA delivery platforms, with particular focus on attempts to create organ-specific vehicles for therapeutic mRNA delivery.

Chimeric RNA-binding protein-based killing switch targeting hepatocellular carcinoma cells.

Cancer cell-specific killing switches are synthetic circuits developed as an intelligent weapon to specifically eliminate malignant cells. RNA-delivered synthetic circuits provide safer means to control oncolytic functions, in which proteolysis-responding capsid-cNOT7 is developed to enable logic computation and modular design. Unfortunately, although circuits containing these capsid-cNOT7s exhibited good performance when introduced as replicons, in modified mRNA (modRNA) delivery, the performance was not quite as good. To improve this situation, alternative modules suitable for modRNA delivery need to be developed. An attractive option is RNA-binding protein (RBP)/riboswitches. In this study, RBPs were engineered by fusing with degron and cleavage sites. The compatibility of these chimeric RBPs with proteolysis-based sensing units were tested. Eight two-input logic gates and four three-input logic gates were implemented. After building this chimeric RBP-based system, we constructed a hepatocellular carcinoma (HCC) cell-specific killing circuit using two proteolysis-based sensing units, a two-input logic OR gate, and a leakproof apoptosis-inducing actuator, which distinguished HCC cells and induced apoptosis in a mixed IMR90-PLC/PRF/5 population.