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1.
Sci Rep ; 12(1): 3794, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735281

ABSTRACT

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.


Subject(s)
Microscopy/methods , SARS-CoV-2/physiology , Virus Internalization , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/transmission , COVID-19/virology , Chlorocebus aethiops , Cytoplasm/chemistry , Cytoplasm/ultrastructure , Cytoplasm/virology , Extracellular Vesicles/chemistry , Extracellular Vesicles/ultrastructure , Giant Cells/chemistry , Giant Cells/physiology , Helium/chemistry , Humans , Ions/chemistry , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
2.
Nat Commun ; 12(1): 6777, 2021 11 22.
Article in English | MEDLINE | ID: covidwho-1528015

ABSTRACT

Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein, reversed-phase ion pair high performance liquid chromatography (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity analytical techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, electrophilic impurities derived from the ionizable cationic lipid component are shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidation and subsequent hydrolysis of the tertiary amine. It thus remains critical to ensure robust analytical methods and stringent manufacturing control to ensure mRNA stability and high activity in LNP delivery systems.


Subject(s)
Drug Delivery Systems , Liposomes/chemistry , Nanoparticles/chemistry , RNA, Messenger/chemistry , Vaccine Potency , Aldehydes/chemistry , Chromatography, Liquid , Humans , Ions/chemistry , Lipids/chemistry , Nucleosides/chemistry , Oxidation-Reduction , Protein Biosynthesis , RNA Stability , /chemistry
3.
J Am Chem Soc ; 143(43): 17975-17982, 2021 11 03.
Article in English | MEDLINE | ID: covidwho-1483092

ABSTRACT

Targeted and efficient delivery of nucleic acids with viral and synthetic vectors is the key step of genetic nanomedicine. The four-component lipid nanoparticle synthetic delivery systems consisting of ionizable lipids, phospholipids, cholesterol, and a PEG-conjugated lipid, assembled by microfluidic or T-tube technology, have been extraordinarily successful for delivery of mRNA to provide Covid-19 vaccines. Recently, we reported a one-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) synthetic delivery system for mRNA relying on amphiphilic Janus dendrimers and glycodendrimers developed in our laboratory. Amphiphilic Janus dendrimers consist of functional hydrophilic dendrons conjugated to hydrophobic dendrons. Co-assembly of IAJDs with mRNA into dendrimersome nanoparticles (DNPs) occurs by simple injection in acetate buffer, rather than by microfluidic devices, and provides a very efficient system for delivery of mRNA to lung. Here we report the replacement of most of the hydrophilic fragment of the dendron from IAJDs, maintaining only its ionizable amine, while changing its interconnecting group to the hydrophobic dendron from amide to ester. The resulting IAJDs demonstrated that protonated ionizable amines play dual roles of hydrophilic fragment and binding ligand for mRNA, changing delivery from lung to spleen and/or liver. Replacing the interconnecting ester with the amide switched the delivery back to lung. Delivery predominantly to liver is favored by pairs of odd and even alkyl groups in the hydrophobic dendron. This simple structural change transformed the targeted delivery of mRNA mediated with IAJDs, from lung to liver and spleen, and expands the utility of DNPs from therapeutics to vaccines.


Subject(s)
Dendrimers/chemistry , RNA, Messenger/chemistry , Amines/chemistry , Animals , Esters/chemistry , Hydrophobic and Hydrophilic Interactions , Ions/chemistry , Mice , Nanoparticles/chemistry , RNA, Messenger/immunology , RNA, Messenger/metabolism , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/immunology , Vaccines, Synthetic/metabolism
4.
Chem Commun (Camb) ; 57(78): 10083-10086, 2021 Sep 30.
Article in English | MEDLINE | ID: covidwho-1404890

ABSTRACT

Zinc deficiency is linked to poor prognosis in COVID-19 patients while clinical trials with zinc demonstrate better clinical outcomes. The molecular targets and mechanistic details of the anti-coronaviral activity of zinc remain obscure. We show that zinc not only inhibits the SARS-CoV-2 main protease (Mpro) with nanomolar affinity, but also viral replication. We present the first crystal structure of the Mpro-Zn2+ complex at 1.9 Å and provide the structural basis of viral replication inhibition. We show that Zn2+ coordinates with the catalytic dyad at the enzyme active site along with two previously unknown water molecules in a tetrahedral geometry to form a stable inhibited Mpro-Zn2+ complex. Further, the natural ionophore quercetin increases the anti-viral potency of Zn2+. As the catalytic dyad is highly conserved across SARS-CoV, MERS-CoV and all variants of SARS-CoV-2, Zn2+ mediated inhibition of Mpro may have wider implications.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , Zinc/chemistry , Animals , Binding Sites , COVID-19/pathology , Catalytic Domain , Chlorocebus aethiops , Coordination Complexes/chemistry , Coordination Complexes/metabolism , Coronavirus 3C Proteases/metabolism , Crystallography, X-Ray , Humans , Ions/chemistry , Kinetics , Molecular Dynamics Simulation , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , SARS-CoV-2/isolation & purification , Surface Plasmon Resonance , Thermodynamics , Vero Cells , Virus Replication/drug effects
5.
Commun Biol ; 4(1): 956, 2021 08 11.
Article in English | MEDLINE | ID: covidwho-1354120

ABSTRACT

Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. Herein, we report a method that can predict the LNP pKa from the structure of the ionizable lipid. We used theoretical, NMR, fluorescent-dye binding, and electrophoretic mobility methods to comprehensively measure protonation of both the ionizable lipid and the formulated LNP. The pKa of the ionizable lipid was 2-3 units higher than the pKa of the LNP primarily due to proton solvation energy differences between the LNP and aqueous medium. We exploited these results to explain a wide range of delivery efficiencies in vitro and in vivo for intramuscular (IM) and intravascular (IV) administration of different ionizable lipids at escalating ionizable lipid-to-mRNA ratios in the LNP. In addition, we determined that more negatively charged LNPs exhibit higher off-target systemic expression of mRNA in the liver following IM administration. This undesirable systemic off-target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP.


Subject(s)
Gene Expression , Ions/chemistry , Lipids/chemistry , Nanoparticles/chemistry , RNA, Messenger/chemistry , RNA, Messenger/genetics , Administration, Intravenous , Animals , Drug Compounding , Humans , Hydrogen-Ion Concentration , Injections, Intramuscular , Mice , Molecular Structure , Nanoparticles/ultrastructure , RNA, Messenger/administration & dosage , RNA, Messenger/pharmacokinetics , Spectrum Analysis , Tissue Distribution , Transfection
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