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mRNA is a new class of drugs that has the potential to revolutionize the treatment of brain tumors. Thanks to the COVID-19 mRNA vaccines and numerous therapy-based clinical trials, it is now clear that lipid nanoparticles (LNPs) are a clinically viable means to deliver RNA therapeutics. However, LNP-mediated mRNA delivery to brain tumors remains elusive. Over the past decade, numerous studies have shown that tumor cells communicate with each other via small extracellular vesicles, which are around 100 nm in diameter and consist of lipid bilayer membrane similar to synthetic lipidbased nanocarriers. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain tumors without undue toxicity. We synthesized LNPs using four components similar to the formulation used in the mRNA COVID19 vaccines (Moderna and Pfizer): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. For the in vitro screen, we tested ten classes of helper lipids based on their abundance in extracellular vesicle membranes, commercial availability, and large-scale production feasibility while keeping rest of the LNP components unchanged. The transfection kinetics of GFP mRNA encapsulated in LNPs and doped with 16 mol% of helper lipids was tested using GL261, U87 and SIM-A9 cell lines. Several LNP formations resulted in stable transfection (upto 5 days) of GFP mRNA in all the cell lines tested in vitro. The successful LNP candidates (enabling >80% transfection efficacy) were then tested in vivo to deliver luciferase mRNA to brain tumors via intrathecal administration in a syngeneic glioblastoma (GBM) mouse model, which confirmed luciferase expression in brain tumors in the cortex. LNPs were then tested to deliver Cre recombinase mRNA in syngeneic GBM mouse model genetically modified to express tdTomato under LoxP marker cassette that enabled identification of LNP targeted cells. mRNA was successfully delivered to tumor cells (70-80% transfected) and a range of different cells in the tumor microenvironment, including tumor-associated macrophages (80-90% transfected), neurons (31- 40% transfected), neural stem cells (39-62% transfected), oligodendrocytes (70-80% transfected) and astrocytes (44-76% transfected). Then, LNP formulations were assessed for delivering Cas9 mRNA and CD81 sgRNA (model protein) in murine syngeneic GBM model to enable gene editing in brain tumor cells. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~94% of brain tumor cells in vivo. In conclusion, we have developed a library of safe LNPs that can transfect GBM cells in vivo with high efficacy. This technology can potentially be used to develop novel mRNA therapies for GBM by delivering single or multiple mRNAs and holds great potential as a tool to study brain tumor biology.
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Delivery of self-amplifying mRNA (SAM) has high potential for infectious disease vaccination due to its self-adjuvanting and dose-sparing properties. Yet a challenge is the susceptibility of SAM to degradation and the need for SAM to reach the cytosol fully intact to enable self-amplification. Lipid nanoparticles are successfully deployed at incredible speed for mRNA vaccination, but aspects such as cold storage, manufacturing, efficiency of delivery, and the therapeutic window can benefit from further improvement. To investigate alternatives to lipid nanoparticles, a class of >200 biodegradable end-capped lipophilic poly(beta-amino ester)s (PBAEs) that enable efficient delivery of SAM in vitro and in vivo as assessed by measuring expression of SAM encoding reporter proteins is developed. The ability of these polymers to deliver SAM intramuscularly in mice is evaluated, and a polymer-based formulation that yields up to 37-fold higher intramuscular (IM) expression of SAM compared to injected naked SAM is identified. Using the same nanoparticle formulation to deliver a SAM encoding rabies virus glycoprotein, the vaccine elicits superior immunogenicity compared to naked SAM delivery, leading to seroconversion in mice at low RNA injection doses. These biodegradable nanomaterials may be useful in the development of next-generation RNA vaccines for infectious diseases.Copyright © 2023 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH.
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Ionizable amino lipids are a major constituent of the lipid nanoparticles for delivering nucleic acid therapeutics (e.g., DLin-MC3-DMA in ONPATTRO , ALC-0315 in Comirnaty , SM-102 in Spikevax ). Scarcity of lipids that are suitable for cell therapy, vaccination, and gene therapies continue to be a problem in advancing many potential diagnostic/therapeutic/vaccine candidates to the clinic. Herein, we describe the development of novel ionizable lipids to be used as functional excipients for designing vehicles for nucleic acid therapeutics/vaccines in vivo or ex vivo use in cell therapy applications. We first studied the transfection efficiency (TE) of LNP-based mRNA formulations of these ionizable lipid candidates in primary human T cells and established a workflow for engineering of primary immune T cells. We then adapted this workflow towards bioengineering of CAR constructs to T cells towards non-viral CAR T therapy. Lipids were also tested in rodents for vaccine applications using self-amplifying RNA (saRNA) encoding various antigens. We have then evaluated various ionizable lipid candidates and their biodistribution along with the mRNA/DNA translation exploration using various LNP compositions. Further, using ionizable lipids from the library, we have shown gene editing of various targets in rodents. We believe that these studies will pave the path to the advancement in nucleic acid based therapeutics and vaccines, or cell gene therapy agents for early diagnosis and detection of cancer, and for targeted genomic medicines towards cancer treatment and diagnosis.
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Cumulative data regardingCOVID-19 infection during pregnancy have demonstrated the ability of SARS-CoV-2 to infect the placenta. However, the mechanisms of SARS-CoV-2 placental viral entry are yet to be defined. SARS-CoV-2 infects cells by binding to the ACE2 receptor. However, SARS-CoV-2 cell entry also requires co-localization of spike protein cleavage by the serine protease TMPRSS2. However, the co-expression of ACE2 and TMPRSS2 in placental cells is debated, raising the question of whether potential non-canonical molecular mechanismsmay be involved in SARS-CoV-2 placental cells' viral entry. Although published data regarding the ability of the SARS-CoV- 2 to infect the fetus are contradicting, the placenta appears to be an immunological barrier to active SARS-CoV-2 infection and vertical transmission;however, the mechanism is unclear. Our experiments demonstrated the ability of the SARS-CoV-2 virus to directly infect the placenta and induce transcriptomic responses in COVID-positive mothers. These transcriptomic responses were characterized by differential expression of specific mRNAs and miRNAs associated with SARS-CoV-2 infection, with induction of specific placental miRNAs that can inhibit viral replication. Failure in such mechanisms may be associated with vertical transmission. Since the start of the COVID-19 pandemic, the COVID-19 mRNA vaccines have been widely used to reduce the morbidity and mortality of SARS-CoV-2 infection. Historically, non-live vaccines have not caused any harm to pregnant mothers;however, it is unclear whether our current understanding of the effects of non-live vaccines serves as a reliable precedent owing to the novel technology used to create these mRNA vaccines. Since there are no definitive data on the possible biodistribution of mRNA vaccines to the placenta, the likelihood of vaccine mRNA reaching the fetus remains uncertain. Little has been reported on the tissue localization of the lipid nanoparticles (LNPs) after intramuscular (IM) administration of the mRNA vaccine. The biodistribution of LNPs containing the mRNA vaccine has been investigated in animal models but not humans. In the murine model, the vaccine LNPs were rapidly disseminated to several organs, including the heart, liver, kidney, lung, and spleen, following IM administration. However, no traditional pharmacokinetic or biodistribution studies have been performed with the mRNA vaccines, including possible biodistribution to breast milk or the placenta.
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Coronavirus disease (COVID-19) is an infectious disease caused by coronavirus. Devel-oping specific drugs for inhibiting replication and viral entry is crucial. Several clinical trial studies are underway to evaluate the efficacy of anti-viral drugs for COVID-19 patients. Nanomedicine formulations can present a novel strategy for targeting the virus life cycle. Nano-drug delivery systems can modify the pharmacodynamics and pharmacokinetics properties of anti-viral drugs and reduce their adverse effects. Moreover, nanocarriers can directly exhibit anti-viral effects. A number of nanocarriers have been studied for this purpose, including liposomes, dendrimers, exosomes and decoy nanoparticles (NPs). Among them, decoy NPs have been considered more as nanodecoys can efficiently protect host cells from the infection of SARS-CoV-2. The aim of this review article is to highlight the probable nanomedicine therapeutic strategies to develop anti-viral drug delivery systems for the treatment of COVID-19.Copyright © 2023 Bentham Science Publishers.
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Rapid developments in the coronavirus disease 2019 (COVID-19) mRNA vaccine showcased the power of lipid nanoparticle (LNP) delivery systems in fighting infectious diseases. In addition, mRNA therapeutics are also in development for cancer immunotherapy. Recently, mRNA therapy has been expanded to induce immune tolerance, the opposite of immune-boosting effects, to treat diseases involving enhanced immune responses including allergies and autoimmune diseases. mRNA LNPs have been used to treat peanut allergy by us and autoimmune experimental autoimmune encephalomyelitis by Ugur Sahin. It is expected that more and more research is going to delve into the immune tolerance field for allergies and autoimmune diseases, where effective therapies are in short supply. © 2023 the author(s), published by De Gruyter, Berlin/Boston.
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Following the recent approval of both siRNA- and mRNA-based therapeutics, nucleic acid therapies are considered a game changer in medicine. Their envisioned widespread use for many therapeutic applications with an array of cellular target sites means that various administration routes will be employed. Concerns exist regarding adverse reactions against the lipid nanoparticles (LNPs) used for mRNA delivery, as PEG coatings on nanoparticles can induce severe antibody-mediated immune reactions, potentially being boosted by the inherently immunogenic nucleic acid cargo. While exhaustive information is available on how physicochemical features of nanoparticles affects immunogenicity, it remains unexplored how the fundamental choice of administration route regulates anti-particle immunity. Here, we directly compared antibody generation against PEGylated mRNA-carrying LNPs administered by the intravenous, intramuscular, or subcutaneous route, using a novel sophisticated assay capable of measuring antibody binding to authentic LNP surfaces with single-particle resolution. Intramuscular injections in mice were found to generate overall low and dose-independent levels of anti-LNP antibodies, while both intravenous and subcutaneous LNP injections generated substantial and highly dose-dependent levels. These findings demonstrate that before LNP-based mRNA medicines can be safely applied to new therapeutic applications, it will be crucial to carefully consider the choice of administration route.
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Lipid nanoparticles as delivery system for mRNA have recently attracted attention to a broader audience as COVID-19 mRNA vaccines. Their low immunogenicity and capability to deliver a variety of nucleic acids renders them an interesting and complementary alternative to gene therapy vectors like AAVs. An important quality attribute of LNPs is the copy number of the encapsulated cargo molecule. This work describes how density and molecular weight distributions obtained by density contrast sedimentation velocity can be used to calculate the mRNA copy number of a degradable lipid nanoparticle formulation. The determined average copy number of 5 mRNA molecules per LNP is consistent with the previous studies using other biophysical techniques, such as single particle imaging microscopy and multi-laser cylindrical illumination confocal spectroscopy (CICS).
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Vaccination has been proved to be the most effective strategy to prevent the Corona Virus Disease 2019 (COVID-19). The mRNA vaccine based on nano drug delivery system (NDDS) - lipid nanoparticles (LNP) has been widely used because of its high effectiveness and safety. Although there have been reports of severe allergic reactions caused by mRNA-LNP vaccines, the mechanism and components of anaphylaxis have not been completely clarified yet. This review focuses on two mRNA-LNP vaccines, BNT162b2 and mRNA-1273. After summarizing the structural characteristics, potential allergens, possible allergic reaction mechanism, and pharmacokinetics of mRNA and LNP in vivo, this article then reviews the evaluation methods for patients with allergic history, as well as the regulations of different countries and regions on people who should not be vaccinated, in order to promote more safe injection of vaccines. LNP has become a recognized highly customizable nucleic acid delivery vector, which not only shows its value in mRNA vaccines, but also has great potential in treating rare diseases, cancers and other broad fields in the future. At the moment when mRNA-LNP vaccines open a new era of nano medicine, it is expected to provide some inspiration for safety research in the process of research, development and evaluation of more nano delivery drugs, and promote more nano drugs successfully to market.Copyright © 2023, Chinese Pharmaceutical Association. All rights reserved.
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Nucleic acids, as a next generation of biotechnology drugs, not only can fundamentally treat diseases, but also own significant platform characteristics in view of technology and production. Therefore, nucleic acid-based drugs have broad clinical applications in biomedical fields. However, nucleic acids are degradable and unstable, and have very low intracellular delivery efficiency in vitro and in vivo, which greatly limits their applications. In recent years, ionizable lipid-based lipid nanoparticles have shown promising application potentials and have been successfully applied to COVID-19 (Coronavirus Disease 2019) vaccines in clinic. Lipid nanoparticles demonstrate high in vivo delivery efficiency and good safety profile due to their unique structural and physicochemical properties, which provides many possibilities for their clinical applications for nucleic acid delivery in the future. This review focused on the characteristics of nucleic acid drugs and their delivery barriers, and discussed the approved nucleic acid drugs to illustrate the key aspects of the success of their delivery carrier system. In addition, problems to be solved in the field were highlighted.Copyright © 2023, Chinese Pharmaceutical Association. All rights reserved.
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Messenger RNA (mRNA) has received great attention in the prevention and treatment of various diseases due to the success of COVID-19 mRNA vaccines (Comirnaty and Spikevax). To meet the therapeutic purpose, it is required that mRNA must enter the target cells and express sufficient proteins. Therefore, the development of effective delivery systems is necessary and crucial. Lipid nanoparticle (LNP) represents a remarkable vehicle that has indeed accelerated mRNA applications in humans, as several mRNA-based therapies have already been approved or are in clinical trials. In this review, we focus on mRNA-LNP mediated anticancer therapy. We summarize the main development strategies of mRNA-LNP formulations, discuss representative therapeutic approaches in cancer, and point out current challenges and possible future directions of this research field. We hope these delivered messages could help further improve the application of mRNA-LNP technology in cancer therapy. This article is protected by copyright. All rights reserved.
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IDWeek is the joint annual meeting of the Infectious Diseases Society of America (IDSA), Society for Healthcare Epidemiology of America (SHEA), the HIV Medicine Association (HIVMA), the Pediatric Infectious Diseases Society (PIDS) and the Society of Infectious Diseases Pharmacists (SIDP). For the first time since the COVID-19 public health emergency began, IDWeek 2022 returned to in-person attendance. It was held in Washington, D.C., and the meeting comprised 5 days of live sessions and on-demand content that included posters and oral presentations.Copyright © 2023 Clarivate.
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Background: Although mRNA SARS-CoV-2 vaccines have received emergencyuse- authorization for infants age 6 months and older, vaccine uptake is slow, stressing that questions of safety and durability of vaccine efficacy remain prominent. Method(s): Infant rhesus macaques (RMs) (n=8/group) at 2 months of age, comparable to human toddler age, were immunized intramuscularly at weeks 0 and 4 with 30mug stabilized prefusion SARS-CoV-2 S-2P spike (S) protein (Washington strain) encoded by mRNA encapsulated in lipid nanoparticles (mRNA-LNP) or 15mug S protein mixed with 3M-052 in stable emulsion (Protein). At 1 year, vaccinated and age-matched unvaccinated RM (n=8) were challenged intranasally (106pfu) and intratracheally (2x106pfu) with B.1.617.2. Lung radiographs and pathology were blindly assessed, viral N gene RNA (vRNA) copies were measured by qPCR in pharyngeal swabs and lung, and neutralizing antibody and peripheral blood T cell responses were measured. Result(s): At 1 year, D614G-specific neutralizing antibody (nAb) titers were still detectable in the Protein (ID50=755;range: 359-1,949) and mRNA-LNP groups (ID50=73;range: 41-240). Both vaccines also induced cross-neutralizing antibodies to B.1.617.2. Peripheral blood CD4+ T cell responses to the ancestral spike protein at week 52 did not differ between the groups. However, median CD8+ T cell responses were higher (p=0.002, Mann Whitney) in the mRNA-LNP group (2.8%;range: 0.9%-7.1%) compared to the Protein group (0.8%;range: 0.1%-1.6%). Control RMs had significantly higher median vRNA copies/ml (1.4+/-2.7x108) in day 4 pharyngeal swabs compared to Protein (3.8+/-6.8x103) or mRNA-LNP (4.4+/-9.7x105) vaccinated RMs. Severe lung pathology was observed in 7 of 8 controls compared to 1 of 8 or 0 of 8 RMs in the mRNA-LNP or Protein group respectively. Protection against lung inflammation was associated with nAb titers (r=-0.592, p=0.003) (Figure 1). Conclusion(s): These results demonstrate that despite lower vaccine doses compared to adults, both protein and mRNA vaccines were safe, induced durable immune responses and provided comparable protective efficacy against infection with a heterologous SARS-CoV-2 variant in infants, implying that early life vaccination of human infants may lead to durable immunity. Neutralizing ID50 antibody titers are a correlate of protection in infant RMs challenged with SARS-CoV-2.
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Vaccination for the prevention of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection is considered the most promising approach to control the pandemic of coronavirus disease 2019 (COVID-19). Although various COVID-19 vaccines have been developed worldwide using several modalities, the vaccines that have shown the highest efficacy to date are mRNA vaccines. Despite their extensive usage, the mechanisms that stimulate the immune responses associated with their immunogenicity and reactogenicity remain largely unknown. In this review, we summarize and discuss current knowledge on immune responses to COVID-19 mRNA vaccines, including potential immune responses and correlating factors underlying the immunogenicity and reactogenicity of mRNA vaccines. We also describe recent trends in the optimization of lipid nanoparticles and vaccination routes. Further understanding of vaccine-elicited immune responses will guide the development of more effective and safe vaccines.
Subject(s)
COVID-19 Vaccines , COVID-19 , Humans , COVID-19 Vaccines/adverse effects , COVID-19/prevention & control , SARS-CoV-2 , RNA, Messenger/genetics , mRNA Vaccines , Antibodies, ViralABSTRACT
Background: In the last two years the pandemic Coronavirus Disease 19 (Covid19), caused by the virus SARS-CoV- 2, described for the first time in Wuhan (China) at the end of 2019, has caused over 359 million cases of infections and 5 million deaths worldwide. To fight this emergency, the pursuit of science has focused on vaccines development against SARS-CoV- 2, including the vaccine BNT162b2. This vaccine contains mRNA translating for SARS-CoV- 2 spike protein wrapped in lipid nanoparticles and its use was approved at the end of 2020. It has been proved that both the BNT162b2 vaccine and the SARS-CoV- 2 infection result in the production of neutralizing antibodies but remains to be clarified the duration of these responses, also versus variants of concern. Method(s): The present study aimed to prospectively analyse and correlate the antibody response and the neutralization capability induced by vaccination with BNT162b2 in a cohort of Sardinian subjects, including a group previously Cov2 infected. Each participant was evaluated for serum SARS-CoV2 Ab IgG RDB, 7 (T1) and 30 (T2) days after the second inoculum of BNT162b2, with chemiluminescent immunoassays (CLIA) and microneutralization assay (MNA) determining the highest serum dilution protecting 90 % of the infected wells. Result(s): All the participants, with or without previous infection, developed a positive antibody response (IgG anti-RBD > 1 AU/ml) within 7 and 30 days from the second vaccine dose and a strong correlation was found between IgG antibody levels and neutralizing activity. A strong difference was observed between the antibody levels of the naive subjects and the ones previously infected, specifically the antibody levels were higher (both at T1 and T2) in the latter group. No significant antibody differences were found for gender and age groups. In addition, there were no significant differences in antibody titre between healthy and immune-mediated subjects. Conclusion(s): In conclusion, this study confirms observed differences in vaccine responses between infection-naive and subjects with history of natural infection, with the presence in the second group of a significantly higher neutralizing and anti-RBD antibody titer. It also demonstrates the strong correlation between anti-RBD antibody titre and neutralizing activity, without significant differences between healthy subjects and subjects with immuno-mediated disease in the short-term. Further follow-up is ongoing in this cohort.
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Vaccine development is among the critical issues for ceasing the COVID-19 pandemic. This review discusses the current usage of biomaterials in vaccine development and provides brief descriptions of the vaccine types and their working mechanisms. New types of vaccine platforms (next-generation vaccines and DNA- or mRNA-based vaccines) are discussed in detail. The mRNA vaccine encoding the spike protein viral antigen can be produced in a cell-free system, suggesting that mRNA vaccines are safer than "classic vaccines" using live or inactivated virus. The mRNA vaccine efficacy is typically high at approximately 95%. However, most mRNA vaccines need to be maintained at −20 or −70 degrees for storage for long periods (half a year) and their transportation because of mRNA vaccine instability in general, although mRNA vaccines with unmodified and self-amplifying RNA (ARCT-154, Arcturus), which have a lyophilized form, have recently been reported to be kept at room temperature. mRNA vaccines are typically entrapped in lipid nanoparticles composed of ionizable lipids, polyethylene glycol (PEG)-lipids, phospholipids, and cholesterol. These components and their composition affect mRNA vaccine stability and efficacy and the size of the mRNA vaccine. The development of an improved mRNA vaccine entrapped in sophisticated biomaterials, such as novel lipid nanoparticles, using new types of biopolymers or lipids is necessary for high efficacy, safe transportation and long-term storage of the next generation of mRNA vaccines under mild conditions. [ FROM AUTHOR] Copyright of Polymer Reviews is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)
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BACKGROUND: Heterologous COVID vaccine priming schedules are immunogenic and effective. This report aims to understand the persistence of immune response to the viral vectored, mRNA and protein-based COVID-19 vaccine platforms used in homologous and heterologous priming combinations, which will inform the choice of vaccine platform in future vaccine development. METHODS: Com-COV2 was a single-blinded trial in which adults ≥ 50 years, previously immunised with single dose 'ChAd' (ChAdOx1 nCoV-19, AZD1222, Vaxzevria, Astrazeneca) or 'BNT' (BNT162b2, tozinameran, Comirnaty, Pfizer/BioNTech), were randomised 1:1:1 to receive a second dose 8-12 weeks later with either the homologous vaccine, or 'Mod' (mRNA-1273, Spikevax, Moderna) or 'NVX' (NVX-CoV2373, Nuvaxovid, Novavax). Immunological follow-up and the secondary objective of safety monitoring were performed over nine months. Analyses of antibody and cellular assays were performed on an intention-to-treat population without evidence of COVID-19 infection at baseline or for the trial duration. FINDINGS: In April/May 2021, 1072 participants were enrolled at a median of 9.4 weeks after receipt of a single dose of ChAd (N = 540, 45% female) or BNT (N = 532, 39% female) as part of the national vaccination programme. In ChAd-primed participants, ChAd/Mod had the highest anti-spike IgG from day 28 through to 6 months, although the heterologous vs homologous geometric mean ratio (GMR) dropped from 9.7 (95% CI (confidence interval): 8.2, 11.5) at D28 to 6.2 (95% CI: 5.0, 7.7) at D196. The heterologous/homologous GMR for ChAd/NVX similarly dropped from 3.0 (95% CI:2.5,3.5) to 2.4 (95% CI:1.9, 3.0). In BNT-primed participants, decay was similar between heterologous and homologous schedules with BNT/Mod inducing the highest anti-spike IgG for the duration of follow-up. The adjusted GMR (aGMR) for BNT/Mod compared with BNT/BNT increased from 1.36 (95% CI: 1.17, 1.58) at D28 to 1.52 (95% CI: 1.21, 1.90) at D196, whilst for BNT/NVX this aGMR was 0.55 (95% CI: 0.47, 0.64) at day 28 and 0.62 (95% CI: 0.49, 0.78) at day 196. Heterologous ChAd-primed schedules produced and maintained the largest T-cell responses until D196. Immunisation with BNT/NVX generated a qualitatively different antibody response to BNT/BNT, with the total IgG significantly lower than BNT/BNT during all follow-up time points, but similar levels of neutralising antibodies. INTERPRETATION: Heterologous ChAd-primed schedules remain more immunogenic over time in comparison to ChAd/ChAd. BNT-primed schedules with a second dose of either mRNA vaccine also remain more immunogenic over time in comparison to BNT/NVX. The emerging data on mixed schedules using the novel vaccine platforms deployed in the COVID-19 pandemic, suggest that heterologous priming schedules might be considered as a viable option sooner in future pandemics. ISRCTN: 27841311 EudraCT:2021-001275-16.
Subject(s)
COVID-19 , Vaccines , Adult , Female , Humans , Male , COVID-19 Vaccines , ChAdOx1 nCoV-19 , BNT162 Vaccine , Pandemics , Single-Blind Method , COVID-19/prevention & control , Vaccination , Immunity , Immunoglobulin G , Antibodies, ViralABSTRACT
Over the past decade, compared to all other macromolecules lipid-based nanocarriers have proven to be an excellent carrier and delivery system for various pharmaceutical drugs of poor bioavailability. In addition to that, they exhibit exceptional qualities such as minimal toxicity, economical scale-up production, great biocompatibility, and high drug loading efficiency. In this study, we have discussed the various types of lipid nanoparticles, such as liposomes, nanostructured lipid carriers, solid lipid nanoparticles, and lipid polymer hybrid nanoparticles. We have also conferred in detail, the composition, shape and size, methods of preparation, advantages, and certain limitations associated with these lipid-based nanocarriers. Additionally, we have exclusively accounted for several examples of lipid-based nanomedicines that have either been approved and commercialized or are under the different phases of clinical trials. The current review overall focuses on the up-to-date research that has recently been published in view of developing lipid-based nanocarriers for various biological applications, including gene therapy, breast cancer therapy, and vaccine development.Copyright © 2022
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It has been proven that mRNA vaccines are highly effective against the COVID-19 outbreak, and low prevalence of side effects has been shown. However, there are still many gaps in our understanding of the biology and biosafety of nucleic acids as components of lipid nanoparticles (LNPs) most often used as a system for inctracellular delivery of mRNA-based vaccines. It is known that LNPs cause severe injection site inflammation, have broad biodistribution profiles, and are found in multiple tissues of the body, including the brain, after administration. The role of new medications with such pharmacokinetics in inflammation developing in inaccessible organs is poorly understood. The study was aimed to assess the effects of various doses of mRNA-LNP expressing the reporter protein (0, 5, 10, and 20 microg of mRNA encoding the firefly luciferase) on the expression of neuroinflammation markers (Tnfalpha, Il1beta, Gfap, Aif1) in the prefrontal cortex and hypothalamus of laboratory animals 4, 8, and 30 h after the intramuscular injection of LNP nanoemulsion. It was shown that mRNA-LNP vaccines in a dose of 10-20 microg of mRNA could enhance Aif1 expression in the hypothalamus 8 h after vaccination, however, no such differences were observed after 30 h. It was found that the Gfap, l11beta, Tnfalpha expression levels in the hypothalamus observed at different times in the experimental groups were different. According to the results, mRNA-LNPs administered by the parenteral route can stimulate temporary activation of microglia in certain time intervals in the dose-dependent and site specific manner.Copyright © 2022 Pirogov Russian National Research Medical University. All rights reserved.
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During the COVID-19 pandemics we have all witnessed the clinical importance of mRNA as current vaccines and future therapeutics. mRNA therapies have a potential to revolutionize cancer treatment. Delivery of mRNA requires lipid nanoparticles (LNP) to protect the cargo from degradation. mRNA has a negative charge and depends on positively charged lipids to be encapsulated in LNP. These lipids can be either ionizable at certain pH or constantly cationic. Even though previous studies had evaluated the formulation properties of ionizable and cationic LNP systems, there is the need to understand their specificity in terms of mRNA delivery and protein expression in breast cancer tumor microenvironment. The objective of this work was to assess the kinetics of LNP cellular uptake and mRNA expression inv breast cancer (BC) cells and fibroblasts, the most frequent cell type in the tumor microenvironment cells, while studying the mechanisms involved in differential behaviors of LNP formulated with cationic and ionizable lipids. To achieve this goal mRNA-LNP containing ionizable lipids (LNP-A) and cationic lipids (LNP-B) were designed and formulated using Nanoassemblr Benchtop microfluidics mixer (Precision NanoSystems). mRNA-LNP were characterized for size, zeta potential using dynamic light scattering (DLS) and mRNA encapsulation efficiency using RiboGreen assay. LNP were tagged with rhodamine lipid to investigate the uptake kinetic and a reporter GFP mRNA to evaluate mRNA expression in murine 4T1 and human MCF7, MDA-231, SUM-159 and T47D breast cancer cells and BJ fibroblasts. Live fluorescence microscopy imaging, IncuCyte S3, was used to determine the LNP uptake and GFP mRNA expression. In vitro biocompatibility was assessed with WST-1 assay. Additionally, expression of mRNA delivered from LNP in tumor microenvironment was evaluated in vivo in a syngeneic 4T1 breast cancer model using mRNA luciferase and IVIS imaging. mRNA-LNPs possessed an average diameter of 77 - 107 nm, narrow size distribution, neutral zeta potential and high mRNA encapsulation efficiency (>94%). Our results demonstrated that mRNA expression was higher in breast cancer cells when delivered from LNP-A formulation and in BJ fibroblasts when delivered from LNP-B. LNP-A, the ionizable LNP, was tested in the breast cancer cells to confirm the efficacy of the delivery. The highest transfection efficacy, from high to low, T-47D, MCF7, SUM-159, 4T1 and MDA-231.We have further investigated the cellular uptake mechanisms of LNP using uptake pathway inhibitors for caveolae endocytosis, clathrin endocytosis, and phagocytosis. Our data confirm that there are differences in mechanisms that govern the uptake of mRNA LNP in breast cancer cells and fibroblasts. Clathrin-mediated endocytosis was active in 4T1 breast cancer cells for ionizable and cationic LNP. Interestingly, despite in vitro differences in uptake and mRNA expression, in vivo results show that both formulations efficiently delivered luciferasemRNA in the tumor microenvironment. Histology results demonstrated similar luciferase expression for both LNP in tumors. Additionally, we were able to confirm the prominent presence of fibroblast and similar distribution in the 4T1 subcutaneous model which could explain the similar efficacy of cationic and ionizable LNP. Understanding uptake and mRNA expression of different LNP formulations in the tumor microenvironment can help in achieving the necessary protein expression for breast cancer therapies. Furthermore, determining the most efficient carrier in early stages may reduce the time required for clinical translation. Acknowledgement: This research was supported in part by CPRIT Core for RNA Therapeutics and Research.