bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2022–05–29
thirteen papers selected by
the Merkel lab, Ludwig-Maximilians University
  1. Tetraethylenepentamine-Coated β Cyclodextrin Nanoparticles for Dual DNA and siRNA Delivery.
  2. Protamine-Lipid-dsRNA Nanoparticles Improve RNAi Efficiency in the Fall Armyworm, Spodoptera frugiperda.
  3. Sulfonium Lipids: Synthesis and Evaluation as DNA Delivery Vectors.
  4. Evaluation of liver specific ionizable lipid nanocarrierin the delivery of siRNA.
  5. Optimization of Lipid Nanoparticles for saRNA Expression and Cellular Activation Using a Design-of-Experiment Approach.
  6. Random Copolymers of Lysine and Isoleucine for Efficient mRNA Delivery.
  7. Interaction of Cationic Carbosilane Dendrimers and Their siRNA Complexes with MCF-7 Cells Cultured in 3D Spheroids.
  8. Rational Design of Bisphosphonate Lipid-like Materials for mRNA Delivery to the Bone Microenvironment.
  9. Inhaled delivery of a lipid nanoparticle encapsulated messenger RNA encoding a ciliary protein for the treatment of primary ciliary dyskinesia.
  10. Glutathione-Sensitive Nanoglue Platform with Effective Nucleic Acids Gluing onto Liposomes for Photo-Gene Therapy.
  11. Effects of inhalable gene transfection as a novel gene therapy for non-small cell lung cancer and malignant pleural mesothelioma.
  12. Bioactive cytomembrane@poly(citrate-peptide)-miRNA365 nanoplatform with immune escape and homologous targeting for colon cancer therapy.
  13. Development of an LNP-Encapsulated mRNA-RBD Vaccine against SARS-CoV-2 and Its Variants.
  1. Pharmaceutics. 2022 Apr 23. pii: 921. [Epub ahead of print]14(5):
    Tetraethylenepentamine-Coated β Cyclodextrin Nanoparticles for Dual DNA and siRNA Delivery.
    Chi-Hsien Liu, Pei-Yin Shih, Cheng-Han Lin, Yi-Jun Chen, Wei-Chi Wu, Chun-Chao Wang.
      Nucleic acid reagents, including plasmid-encoded genes and small interfering RNA (siRNA), are promising tools for validating gene function and for the development of therapeutic agents. Native β-cyclodextrins (BCDs) have limited efficiency in gene delivery due to their instable complexes with nucleic acid. We hypothesized that cationic BCD nanoparticles could be an efficient carrier for both DNA and siRNA. Tetraethylenepentamine-coated β-cyclodextrin (TEPA-BCD) nanoparticles were synthesized, characterized, and evaluated for targeted cell delivery of plasmid DNA and siRNA. The cationic TEPA coating provided ideal zeta potential and effective nucleic acid binding ability. When transfecting plasmid encoding green fluorescent protein (GFP) by TEPA-BCD, excellent GFP expression could be achieved in multiple cell lines. In addition, siRNA transfected by TEPA-BCD suppressed target GFP gene expression. We showed that TEPA-BCD internalization was mediated by energy-dependent endocytosis via both clathrin-dependent and caveolin-dependent endocytic pathways. TEPA-BCD nanoparticles provide an effective means of nucleic acid delivery and can act as potential carriers in future pharmaceutical application.
    Keywords:  knockdown; nanoparticle; siRNA; transfection; β-cyclodextrin
    DOI:  https://doi.org/10.3390/pharmaceutics14050921
  2. J Agric Food Chem. 2022 May 25.
    Protamine-Lipid-dsRNA Nanoparticles Improve RNAi Efficiency in the Fall Armyworm, Spodoptera frugiperda.
    Ramesh Kumar Dhandapani, Dhandapani Gurusamy, Subba Reddy Palli.
      Developing safe and effective double-stranded RNA (dsRNA) delivery systems remains a major challenge for gene silencing, especially in lepidopteran insects. This study evaluated the protamine sulfate (PS)/lipid/dsRNA nanoparticle (NP) delivery system for RNA interference (RNAi) in cells and larvae of the fall armyworm (FAW), Spodoptera frugiperda, a major worldwide pest. A highly efficient gene delivery formulation was prepared using a cationic biopolymer, PS, and a cationic lipid, Cellfectin (CF), complexed with dsRNA. The NPs were prepared by a two-step self-assembly method. The formation of NPs was revealed by dynamic light scattering and transmission electron microscopy. The formation of CF/dsRNA/PS NPs was spherical in shape and size, ranging from 20 to 100 nm with a positive charge (+23.3 mV). Interestingly, prepared CF/dsRNA/PS NPs could protect dsRNA (95%) from nuclease degradation and thus significantly improve the stability of dsRNA. Formulations prepared by combining EGFP DNA with CF/PS increased transfection efficiency in Sf9 cells compared to PS/EGFP and CF/EGFP NPs. Also, the PS/CF/dsRNA NPs enhanced the endosomal escape for the intracellular delivery of dsRNA. The gene knockdown efficiency was assessed in Sf9 Luciferase (Luc) stable cells after a 72 h incubation with CF/dsRNA/PS, PS/dsRNA, CF/dsRNA, or naked dsRNA. Knockdown of the Luc gene was detected in CF/dsRNA/PS (76%) and PS/dsRNA (42.4%) not CF/dsRNA (19.5%) and naked dsRNA (10.3%) in Sf9 Luc cells. Moreover, CF/dsIAP/PS (25 μg of dsRNA targeting the inhibitor of apoptosis, IAP, gene of FAW) NPs showed knockdown of the IAP gene (39.5%) and mortality (55%) in FAW larvae. These results highlight the potential application of PS/lipid/dsRNA NPs for RNA-mediated control of insect pests.
    Keywords:  Cellfectin; EGFP transfection; RNA interference; Sf9 cells; Spodoptera frugiperda; double-stranded RNA; protamine
    DOI:  https://doi.org/10.1021/acs.jafc.2c00901
  3. Curr Drug Deliv. 2022 May 19.
    Sulfonium Lipids: Synthesis and Evaluation as DNA Delivery Vectors.
    Jing Li, Lei Zhang, Yanjie Lu, Yue Lin, Kun Yang, Xiaodong Zhou, Guinan Shen.
       BACKGROUND: Cationic lipids can be used as nonviral vectors in gene delivery therapy. Most cationic lipids contain quaternary ammonium that can bond to negative phosphates of the plasmid. In this study, sulfonium-a trialkylated sulfur cation was adopted in the synthesis of a series of cationic lipids which were evaluated ability as gene delivery vectors.
    METHODS: The sulfonium lipids were synthesized by condensing cyclic thioether and aliphatic carbon chains with ethoxy linkage and characterized the structure by NMR and mass. The DNA condensing abilities of sulfonium lipids were evaluated using a gel retardation experiment. Sulfonium lipids/DNA condensates were measured for particle size and Zeta potential. The cytotoxicity of sulfoniums was evaluated with MTT assay. The intracellular uptakes of sulfonium lipid/DNA complexes were observed with a fluorescence microscope.
    RESULTS: The results showed that the sulfonium head can effectively bond to the phosphate of DNA. When S/P ratio is larger than 10/1, sulfonium lipids with longer carbon chains can completely condense DNA to form a nanoparticle with particle size ranging from 135 nm to 155 nm and zeta potential ranging from 28 mV to 42 mV. The IC50 of sulfonium lipids on HepG2 cells ranged from 2.37 μg/mL to 3.67 μg/mL. Cellular uptake experiments showed that sulfonium lipids/DNA condensate can be taken into cells.
    CONCLUSION: Sulfonium lipids can effectively condense DNA and transfer DNA into cells. The sulfonium compound is worth of further development to reduce the cytotoxicity and increase transfection rate as gene vectors.
    Keywords:  Cationic Lipids; DNA; Gene Delivery; Non-viral Vector; Quaternary Ammonium.; Sulfonium
    DOI:  https://doi.org/10.2174/1567201819666220519122622
  4. Chem Phys Lipids. 2022 May 24. pii: S0009-3084(22)00035-4. [Epub ahead of print] 105207
    Evaluation of liver specific ionizable lipid nanocarrierin the delivery of siRNA.
    Shilpa Rana, Archana Bhatnagar, Suman Singh, Nirmal Prabhakar.
      Hepcidin, a key regulator of iron homeostasis, has been implicated in the pathogenesis of various iron-related diseases. Although small interfering RNA (siRNA) are potent to modulate the expression of hepcidin, their bioavailability remains a major issue. The β-galactopyranoside-conjugated liposomes (GAL-liposome) targeting liver synthesized hepcidin were prepared by thin lipid film hydration method to encapsulate siRNA and the conjugation of β-galactopyranoside to the lipid nanocarrier was achieved by covalent chemistry. The prepared siRNA loaded GAL-lip were spherical with around 50 nm radius in size as observed by HR-TEM. The zeta potential and polydispersity index of the prepared liposomes were -19.9±0.96 mV and 0.44±0.05, respectively. The encapsulation efficiency as determined by dialysis bag method was around 91.76±1.74%. The cell viability and cellular uptake analysis was examined in HepG2 cells by MTT assay and flow cytometry, respectively. The stability and cumulative release of siRNA was also assessed. The hepcidin mRNA expression on administration of siRNA loaded GAL-lip was determined in HepG2 cells and in lipopolysaccharide-induced mice model followed by examining itsin vivo biodistribution by fluorescence microscopy. The results suggested thatsiRNA loaded GAL-lip reduced the hepcidin levels, thus, highlighting a novel ligand conjugated ionizable lipid-based nanocarrier for inducing RNA interference.
    Keywords:  asialoglycoprotein receptors; hepcidin siRNA; liposomes; liver; β-D-galactopyranosyl
    DOI:  https://doi.org/10.1016/j.chemphyslip.2022.105207
  5. Mol Pharm. 2022 May 23.
    Optimization of Lipid Nanoparticles for saRNA Expression and Cellular Activation Using a Design-of-Experiment Approach.
    Han Han Ly, Simon Daniel, Shekinah K V Soriano, Zoltán Kis, Anna K Blakney.
      Lipid nanoparticles (LNPs) are the leading technology for RNA delivery, given the success of the Pfizer/BioNTech and Moderna COVID-19 mRNA (mRNA) vaccines, and small interfering RNA (siRNA) therapies (patisiran). However, optimization of LNP process parameters and compositions for larger RNA payloads such as self-amplifying RNA (saRNA), which can have complex secondary structures, have not been carried out. Furthermore, the interactions between process parameters, critical quality attributes (CQAs), and function, such as protein expression and cellular activation, are not well understood. Here, we used two iterations of design of experiments (DoE) (definitive screening design and Box-Behnken design) to optimize saRNA formulations using the leading, FDA-approved ionizable lipids (MC3, ALC-0315, and SM-102). We observed that PEG is required to preserve the CQAs and that saRNA is more challenging to encapsulate and preserve than mRNA. We identified three formulations to minimize cellular activation, maximize cellular activation, or meet a CQA profile while maximizing protein expression. The significant parameters and design of the response surface modeling and multiple response optimization may be useful for designing formulations for a range of applications, such as vaccines or protein replacement therapies, for larger RNA cargoes.
    Keywords:  Box−Behnken Design; cytokine response; definitive screening design; design-of-experiment (DoE); lipid nanoparticle (LNP); mRNA (mRNA); protein expression; self-amplifying mRNA (saRNA)
    DOI:  https://doi.org/10.1021/acs.molpharmaceut.2c00032
  6. Int J Mol Sci. 2022 May 11. pii: 5363. [Epub ahead of print]23(10):
    Random Copolymers of Lysine and Isoleucine for Efficient mRNA Delivery.
    Iuliia Pilipenko, Olga Korovkina, Nina Gubina, Viktoria Ekimova, Anastasia Ishutinova, Evgenia Korzhikova-Vlakh, Tatiana Tennikova, Viktor Korzhikov-Vlakh.
      Messenger RNA (mRNA) is currently of great interest as a new category of therapeutic agent, which could be used for prevention or treatment of various diseases. For this mRNA requires effective delivery systems that will protect it from degradation, as well as allow cellular uptake and mRNA release. Random poly(lysine-co-isoleucine) polypeptides were synthesized and investigated as possible carriers for mRNA delivery. The polypeptides obtained under lysine:isoleucine monomer ratio equal to 80/20 were shown to give polyplexes with smaller size, positive ζ-potential and more than 90% encapsulation efficacy. The phase inversion method was proposed as best way for encapsulation of mRNA into polyplexes, which are based on obtained amphiphilic copolymers. These copolymers showed efficacy in protection of bound mRNA towards ribonuclease and lower toxicity as compared to lysine homopolymer. The poly(lysine-co-isoleucine) polypeptides showed greater than poly(ethyleneimine) efficacy as vectors for transfection of cells with green fluorescent protein and firefly luciferase encoding mRNAs. This allows us to consider obtained copolymers as promising candidates for mRNA delivery applications.
    Keywords:  cationic polypeptides; mRNA delivery; polyplexes; random amphiphilic copolymers; transfection
    DOI:  https://doi.org/10.3390/ijms23105363
  7. Cells. 2022 May 19. pii: 1697. [Epub ahead of print]11(10):
    Interaction of Cationic Carbosilane Dendrimers and Their siRNA Complexes with MCF-7 Cells Cultured in 3D Spheroids.
    Kamila Białkowska, Piotr Komorowski, Rafael Gomez-Ramirez, Francisco Javier de la Mata, Maria Bryszewska, Katarzyna Miłowska.
      Cationic dendrimers are effective carriers for the delivery of siRNA into cells; they can penetrate cell membranes and protect nucleic acids against RNase degradation. Two types of dendrimers (CBD-1 and CBD-2) and their complexes with pro-apoptotic siRNA (Mcl-1 and Bcl-2) were tested on MCF-7 cells cultured as spheroids. Cytotoxicity of dendrimers and dendriplexes was measured using the live-dead test and Annexin V-FITC Apoptosis Detection Kit (flow cytometry). Uptake of dendriplexes was examined using flow cytometry and confocal microscopy. The live-dead test showed that for cells in 3D, CBD-2 is more toxic than CBD-1, contrasting with the data for 2D cultures. Attaching siRNA to a dendrimer molecule did not lead to increased cytotoxic effect in cells, either after 24 or 48 h. Measurements of apoptosis did not show a high increase in the level of the apoptosis marker after 24 h exposure of spheroids to CBD-2 and its dendriplexes. Measurements of the internalization of dendriplexes and microscopy images confirmed that the dendriplexes were transported into cells of the spheroids. Flow cytometry analysis of internalization indicated that CBD-2 transported siRNAs more effectively than CBD-1. Cytotoxic effects were visible after incubation with 3 doses of complexes for CBD-1 and both siRNAs.
    Keywords:  3D cell culture; carbosilane dendrimers; dendriplexes; nanocarriers; siRNA; spheroids
    DOI:  https://doi.org/10.3390/cells11101697
  8. J Am Chem Soc. 2022 May 26.
    Rational Design of Bisphosphonate Lipid-like Materials for mRNA Delivery to the Bone Microenvironment.
    Lulu Xue, Ningqiang Gong, Sarah J Shepherd, Xinhong Xiong, Xueyang Liao, Xuexiang Han, Gan Zhao, Chao Song, Xisha Huang, Hanwen Zhang, Marshall S Padilla, Jingya Qin, Yi Shi, Mohamad-Gabriel Alameh, Darrin J Pochan, Karin Wang, Fanxin Long, Drew Weissman, Michael J Mitchell.
      The development of lipid nanoparticle (LNP) formulations for targeting the bone microenvironment holds significant potential for nucleic acid therapeutic applications including bone regeneration, cancer, and hematopoietic stem cell therapies. However, therapeutic delivery to bone remains a significant challenge due to several biological barriers, such as low blood flow in bone, blood-bone marrow barriers, and low affinity between drugs and bone minerals, which leads to unfavorable therapeutic dosages in the bone microenvironment. Here, we construct a series of bisphosphonate (BP) lipid-like materials possessing a high affinity for bone minerals, as a means to overcome biological barriers to deliver mRNA therapeutics efficiently to the bone microenvironment in vivo. Following in vitro screening of BP lipid-like materials formulated into LNPs, we identified a lead BP-LNP formulation, 490BP-C14, with enhanced mRNA expression and localization in the bone microenvironment of mice in vivo compared to 490-C14 LNPs in the absence of BPs. Moreover, BP-LNPs enhanced mRNA delivery and secretion of therapeutic bone morphogenetic protein-2 from the bone microenvironment upon intravenous administration. These results demonstrate the potential of BP-LNPs for delivery to the bone microenvironment, which could potentially be utilized for a range of mRNA therapeutic applications including regenerative medicine, protein replacement, and gene editing therapies.
    DOI:  https://doi.org/10.1021/jacs.2c02706
  9. Pulm Pharmacol Ther. 2022 May 22. pii: S1094-5539(22)00025-6. [Epub ahead of print] 102134
    Inhaled delivery of a lipid nanoparticle encapsulated messenger RNA encoding a ciliary protein for the treatment of primary ciliary dyskinesia.
    Caroline J Woo, Ayed Allawzi, Nicholas Clark, Neha Kaushal, Tim Efthymiou, Maike Thamsen, Jane Ngyuen, Richard Wooster, James Sullivan.
      Primary ciliary dyskinesia (PCD) is a respiratory disease caused by dysfunction of the cilia with currently no approved treatments. This predominantly autosomal recessive disease is caused by mutations in any one of over 50 genes involved in cilia function; DNAI1 is one of the more frequently mutated genes, accounting for approximately 5-10% of diagnosed PCD cases. A codon-optimized mRNA encoding DNAI1 and encapsulated in a lipid nanoparticle (LNP) was administered to mice via aerosolized inhalation resulting in the expression human DNAI1 in the multiciliated cells of the pseudostratified columnar epithelia. The spatial localization of DNAI1 expression in the bronchioles indicate that delivery of the DNAI1 mRNA transpires the lower airways. In a PCD disease model, exposure to the LNP-encapsulated DNAI1 mRNA resulted in increased ciliary beat frequency using high speed videomicroscopy showing the potential for an mRNA therapeutic to correct cilia function in patients with PCD due to DNAI1 mutations.
    Keywords:  Dynein axonemal intermediate chain 1; Inhaled; Lipid nanoparticle; Messenger RNA; Primary ciliary dyskinesia
    DOI:  https://doi.org/10.1016/j.pupt.2022.102134
  10. ACS Appl Mater Interfaces. 2022 May 24.
    Glutathione-Sensitive Nanoglue Platform with Effective Nucleic Acids Gluing onto Liposomes for Photo-Gene Therapy.
    Yue Yu, Zhenfeng Wang, Sichen Wu, Chunmeng Zhu, Xianshe Meng, Chao Li, Sheng Cheng, Wei Tao, Feng Wang.
      Liposomal spherical nucleic acids possess a high density of nucleic acids, e.g., DNA, on a liposomal core. There are two approaches to conjugate DNA onto the zwitterionic liposomes, i.e., covalent and noncovalent conjugation, otherwise using cationic liposomes. However, complex and expensive DNA chemical modification methods need to seek a novel and easy-operating approach to decorating DNA onto liposomes. Inspired by the nanoparticle solution as nanoglues for gels and biological tissues, we use MnO2 nanosheets to "glue" DNA onto liposomes without DNA modification. In tumor cells with a high glutathione concentration, MnO2-based nanoglues are degraded, generating water-soluble Mn2+ ions, further "unglue" DNA (i.e., DNAzyme), and liposomes. Using the intelligent liposomal nanoglue (DNAzyme/MnO2/Lip) combining glutathione-sensitive MnO2 nanosheets, gene silencing agent DNAzyme, and photosensitizer Chlorin e6 (Ce6) in liposomes, effective photo-gene therapy was demonstrated.
    Keywords:  MnO2; liposome; nanoglue; nucleic acid; photo-gene therapy
    DOI:  https://doi.org/10.1021/acsami.2c04022
  11. Sci Rep. 2022 May 23. 12(1): 8634
    Effects of inhalable gene transfection as a novel gene therapy for non-small cell lung cancer and malignant pleural mesothelioma.
    Misa Ichikawa, Naomi Muramatsu, Wataru Matsunaga, Takahiro Ishikawa, Tomoyuki Okuda, Hirokazu Okamoto, Akinobu Gotoh.
      Gene therapy using vectors has attracted attention in recent years for the treatment of cancers caused by gene mutations. Besides, new treatments are imperative for lung cancer, including non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM), due to its high mortality. We developed a minimally invasive and orally inhalable tumor suppressor gene drug (SFD-p16 and SFD-p53) with non-viral vectors for lung cancer treatment by combining tumor suppressor genes with an inhalant powder that can deliver active ingredients directly to the lung. We used NSCLC (A549 and H1299) and MPM (H2052) cell lines in an air-liquid interface culture. Transfection of A549 and H2052 cells with SFD-p16 significantly increased p16 mRNA expression levels and decreased cell proliferation in both cell lines. Similar results were obtained with transfection of H1299 with the inhalable gene drug SFD-p53. In an in vivo experiment, a mouse model of lung cancer with orthotopically transplanted luciferase-expressing A549 cells was subjected to intratracheal insufflation of SFD-p16. Consequently, SFD-p16 effectively and directly affected lung cancer. This study suggests that inhalable gene drugs are effective treatments for NSCLC and MPM. We expect inhalable gene drugs to present a novel gene therapy agent for lung cancer that patients can self-administer.
    DOI:  https://doi.org/10.1038/s41598-022-12624-4
  12. Mater Today Bio. 2022 Jun;15 100294
    Bioactive cytomembrane@poly(citrate-peptide)-miRNA365 nanoplatform with immune escape and homologous targeting for colon cancer therapy.
    Long Zhang, Wan Zhang, Hang Peng, Tianli Shen, Min Wang, Meng Luo, Xiaoyan Qu, Fengyi Qu, Wenguang Liu, Bo Lei, Shuanying Yang.
      Colon cancer is one of the most common gastrointestinal tumors in the world. Currently, the commonly used methods such as radiotherapy, chemotherapy and drug treatments are often ineffective and have significant side effects. Here we developed a safe and efficient biomaterials based anti-tumor nanoplatform (M@NPs/miR365), which was formed with poly (citrate-peptide) (PCP), miRNA365 mimic and MC38 cancer cell membrane (M). PCP could efficiently deliver miR365 mimic into MC38 cancer cells, promote the apoptosis of MC38 tumor cells and regulate the expression of Bcl2 and Ki67 in vitro. Tumor cell membranes were prepared by a fast and convenient sonication method. This tumor cell membrane-coated drug delivery system M@NPs can effectively reduce macrophage uptake and increase the stability of NPs. And the MC38 tumor model mice experiment showed that M@NPs/miR365 via caudal vein injection effectively inhibit tumor development. Based on the immune escape and homologous targeting of cancer cells and efficient gene transfection ability of NPs, this "Trojan horse" like "Pseudotumor cell" carries the target gene miR365 mimic to the tumor site and realizes cancer therapy. Noteworthy, the drug delivery system has good biocompatibility. Thus, this safe drug delivery strategy mediated by cancer cell membrane and gene therapy may have a certain significance for reducing the gap between nanoplatform and tumor clinical treatment.
    Keywords:  Bioactive biomaterials; Colon cancer therapy; Gene delivery; Polycitrate; Pseudotumor cell
    DOI:  https://doi.org/10.1016/j.mtbio.2022.100294
  13. Pharmaceutics. 2022 May 20. pii: 1101. [Epub ahead of print]14(5):
    Development of an LNP-Encapsulated mRNA-RBD Vaccine against SARS-CoV-2 and Its Variants.
    Cong Liu, Nino Rcheulishvili, Zhigao Shen, Dimitri Papukashvili, Fengfei Xie, Ziqian Wang, Xingyun Wang, Yunjiao He, Peng George Wang.
      Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is undoubtedly the most challenging pandemic in the current century and remains a global health emergency. As the number of COVID-19 cases in the world is on the rise and variants continue to emerge, there is an urgent need for vaccines. Among all immunization approaches, mRNA vaccines have demonstrated more promising results in response to this challenge. Herein, we designed an mRNA-based vaccine encoding the receptor-binding domain (RBD) of SARS-CoV-2 encapsulated in lipid nanoparticles (LNPs). Intramuscular (i.m.) administration of the mRNA-RBD vaccine elicited broad-spectrum neutralizing antibodies and cellular responses against not only the wild-type SARS-CoV-2 virus but also Delta and Omicron variants. These results indicated that two doses of mRNA-RBD immunization conferred a strong immune response in mice against the wild-type SARS-CoV-2, while the booster dose provided a sufficient immunity against SARS-CoV-2 and its variants. Taken together, the three-dose regimen strategy of the mRNA-RBD vaccine proposed in the present study appears to be a promising reference for the development of mRNA vaccines targeting SARS-CoV-2 variants.
    Keywords:  COVID-19; Delta; LNPs; Omicron; RBD; SARS-CoV-2; mRNA vaccine; neutralizing antibodies
    DOI:  https://doi.org/10.3390/pharmaceutics14051101
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