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1.
Biomed Pharmacother ; 145: 112385, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1565522

ABSTRACT

Chemically modified mRNA represents a unique, efficient, and straightforward approach to produce a class of biopharmaceutical agents. It has been already approved as a vaccination-based method for targeting SARS-CoV-2 virus. The COVID-19 pandemic has highlighted the prospect of synthetic modified mRNA to efficiently and safely combat various diseases. Recently, various optimization advances have been adopted to overcome the limitations associated with conventional gene therapeutics leading to wide-ranging applications in different disease conditions. This review sheds light on emerging directions of chemically modified mRNAs to prevent and treat widespread chronic diseases, including metabolic disorders, cancer vaccination and immunotherapy, musculoskeletal disorders, respiratory conditions, cardiovascular diseases, and liver diseases.


Subject(s)
COVID-19/prevention & control , Chronic Disease/prevention & control , Chronic Disease/therapy , Genetic Therapy/methods , Immunotherapy/methods , Pandemics/prevention & control , RNA, Messenger/chemistry , SARS-CoV-2/immunology , Vaccines, Synthetic , Biological Availability , Drug Carriers , Forecasting , Gene Transfer Techniques , Genetic Vectors/administration & dosage , Genetic Vectors/therapeutic use , Humans , Immunotherapy, Active , RNA Stability , RNA, Messenger/administration & dosage , RNA, Messenger/immunology , RNA, Messenger/therapeutic use , SARS-CoV-2/genetics , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , /immunology
2.
J Phys Chem Lett ; 12(45): 11199-11205, 2021 Nov 18.
Article in English | MEDLINE | ID: covidwho-1510547

ABSTRACT

Recent advances in RNA-based medicine have provided new opportunities for the global current challenge, i.e., the COVID-19 pandemic. Novel vaccines are based on a messenger RNA (mRNA) motif with a lipid nanoparticle (LNP) vector, consisting of high content of unique pH-sensitive ionizable lipids (ILs). Here we provide molecular insights into the role of the ILs and lipid mixtures used in current mRNA vaccines. We observed that the lipid mixtures adopted a nonlamellar organization, with ILs separating into a very disordered, pH-sensitive phase. We describe structural differences of the two ILs leading to their different congregation, with implications for the vaccine stability. Finally, as RNA interacts preferentially with IL-rich phases located at the regions with high curvature of lipid phase, local changes in RNA flexibility and base pairing are induced by lipids. A proper atomistic understanding of RNA-lipid interactions may enable rational tailoring of LNP composition for efficient RNA delivery.


Subject(s)
COVID-19 Vaccines/chemistry , Lipids/chemistry , RNA, Messenger/chemistry , Humans , Lipid Bilayers/chemistry , Models, Molecular , Molecular Dynamics Simulation
3.
J Endocrinol Invest ; 44(12): 2675-2684, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1504521

ABSTRACT

PURPOSE: Due to relevant repercussions on reproductive medicine, we aimed to evaluate feasibility of RT-PCR as a detection method of SARS-CoV-2 RNA in seminal fluid. METHODS: A qualitative determination of the RT-PCR assays in semen was performed through different approaches: (1) efficiency of RNA extraction from sperm and seminal plasma was determined using PRM1 and PRM2 mRNA and a heterologous system as control; (2) samples obtained by diluting viral preparation from a SARS-CoV-2 panel (virus cultured in Vero E6 cell lines) were tested; (3) viral presence in different fractions of seminal fluid (whole sample, seminal plasma and post-centrifugation pellet) was evaluated. Semen samples from mild and recovered COVID-19 subjects were collected by patients referring to the Infectious Disease Department of the Policlinico Umberto I Hospital - "Sapienza" University of Rome. Control subjects were recruited at the Laboratory of Seminology-Sperm Bank "Loredana Gandini'' of the same hospital. RESULTS: The control panel using viral preparations diluted in saline and seminal fluid showed the capability to detect viral RNA presence with Ct values depending on the initial viral concentration. All tested semen samples were negative for SARS-CoV-2, regardless of the nasopharyngeal swab result or seminal fluid fraction. CONCLUSION: These preliminary data show that RT-PCR for SARS-CoV-2 RNA testing appears to be a feasible method for the molecular diagnosis of SARS-CoV-2 in seminal fluid, supported by results of the control panel. The ability to detect SARS-CoV-2 in semen is extremely important for reproductive medicine, especially in assisted reproductive technology and sperm cryopreservation.


Subject(s)
COVID-19/diagnosis , Pathology, Molecular/methods , Semen/virology , Adult , Animals , Chlorocebus aethiops , Feasibility Studies , Humans , Male , RNA, Messenger/chemistry , RNA, Viral/chemistry , Real-Time Polymerase Chain Reaction , Reproductive Techniques , Vero Cells
4.
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
5.
Acc Chem Res ; 54(21): 4001-4011, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1475239

ABSTRACT

Since the U.S. Food and Drug Administration (FDA) granted emergency use authorization for two mRNA vaccines against SARS-CoV-2, mRNA-based technology has attracted broad attention from the scientific community to investors. When delivered intracellularly, mRNA has the ability to produce various therapeutic proteins, enabling the treatment of a variety of illnesses, including but not limited to infectious diseases, cancers, and genetic diseases. Accordingly, mRNA holds significant therapeutic potential and provides a promising means to target historically hard-to-treat diseases. Current clinical efforts harnessing mRNA-based technology are focused on vaccination, cancer immunotherapy, protein replacement therapy, and genome editing. The clinical translation of mRNA-based technology has been made possible by leveraging nanoparticle delivery methods. However, the application of mRNA for therapeutic purposes is still challenged by the need for specific, efficient, and safe delivery systems.This Account highlights key advances in designing and developing combinatorial synthetic lipid nanoparticles (LNPs) with distinct chemical structures and properties for in vitro and in vivo intracellular mRNA delivery. LNPs represent the most advanced nonviral nanoparticle delivery systems that have been extensively investigated for nucleic acid delivery. The aforementioned COVID-19 mRNA vaccines and one LNP-based small interfering RNA (siRNA) drug (ONPATTRO) have received clinical approval from the FDA, highlighting the success of synthetic ionizable lipids for in vivo nucleic acid delivery. In this Account, we first summarize the research efforts from our group on the development of bioreducible and biodegradable LNPs by leveraging the combinatorial chemistry strategy, such as the Michael addition reaction, which allows us to easily generate a large set of lipidoids with diverse chemical structures. Next, we discuss the utilization of a library screening strategy to identify optimal LNPs for targeted mRNA delivery and showcase the applications of the optimized LNPs in cell engineering and genome editing. Finally, we outline key challenges to the clinical translation of mRNA-based therapies and propose an outlook for future directions of the chemical design and optimization of LNPs to improve the safety and specificity of mRNA drugs. We hope this Account provides insight into the rational design of LNPs for facilitating the development of mRNA therapeutics, a transformative technology that promises to revolutionize future medicine.


Subject(s)
COVID-19 Vaccines/pharmacology , Gene Editing , Gene Transfer Techniques , Lipids/chemistry , Nanoparticles/chemistry , RNA, Messenger/pharmacology , COVID-19/drug therapy , COVID-19 Vaccines/chemistry , Genetic Therapy , Humans , RNA, Messenger/chemistry , SARS-CoV-2/drug effects
6.
Nucleic Acid Ther ; 31(5): 321-323, 2021 10.
Article in English | MEDLINE | ID: covidwho-1467290

ABSTRACT

The utilization of the mRNA-based Pfizer-BioNTech and Moderna coronavirus disease 2019 (COVID-19) vaccines represents the culmination of many years of nonviral nucleic acid delivery, but more importantly, they signify a massive clinical scientific success. Scientists working in the area of nucleic acid delivery using lipid nanoparticles will undoubtedly be energized by the success of these vaccines and begin to collect much needed data in the realm of nonviral-based RNA and DNA delivery, specifically, the use of lipid nanoparticles, the immune response, safety, and efficacy. It is easily conceivable that in the future we can utilize these data to help streamline our approach for the delivery of DNA for gene therapy and regulatory RNAs for therapeutic and regenerative medicine (ie, wound repair) applications.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , DNA/pharmacokinetics , Gene Transfer Techniques , RNA, Messenger/pharmacokinetics , Biotechnology/trends , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/biosynthesis , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/genetics , DNA/chemistry , Data Mining , Dependovirus/genetics , Dependovirus/immunology , Humans , Liposomes/chemistry , Liposomes/pharmacokinetics , Nanoparticles/administration & dosage , Nanoparticles/chemistry , RNA, Messenger/chemistry , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity
7.
Eur J Med Chem ; 227: 113910, 2022 Jan 05.
Article in English | MEDLINE | ID: covidwho-1458683

ABSTRACT

The current COVID-19 epidemic has greatly accelerated the application of mRNA technology to our real world, and during this battle mRNA has proven it's unique advantages compared to traditional biopharmaceutical and vaccine technology. In order to overcome mRNA instability in human physiological environments, mRNA chemical modifications and nano delivery systems are two key factors for their in vivo applications. In this review, we would like to summarize the challenges for clinical translation of mRNA-based therapeutics, with an emphasis on recent advances in innovative materials and delivery strategies. The nano delivery systems include lipid delivery systems (lipid nanoparticles and liposomes), polymer complexes, micelles, cationic peptides and so on. The similarities and differences of lipid nanoparticles and liposomes are also discussed. In addition, this review also present the applications of mRNA to other areas than COVID-19 vaccine, such as infectious diseases, tumors, and cardiovascular disease, for which a variety of candidate vaccines or drugs have entered clinical trials. Furthermore, mRNA was found that it might be used to treat some genetic disease, overcome the immaturity of the immune system due to the small fetal size in utero, treat some neurological diseases that are difficult to be treated surgically, even be used in advancing the translation of iPSC technology et al. In short, mRNA has a wide range of applications, and its era has just begun.


Subject(s)
/chemistry , RNA, Messenger/chemistry , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/chemistry , Humans , Liposomes/chemistry , Micelles , Nanoparticles/chemistry , Peptides/chemistry , RNA, Messenger/metabolism , SARS-CoV-2/isolation & purification
8.
Cell ; 184(21): 5271-5274, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1433036

ABSTRACT

This year's Lasker∼Debakey Clinical Research Award honors Katalin Karikó and Drew Weissman for the development of a therapeutic technology based on nucleoside-modification of messenger RNA, enabling the rapid development of the highly effective COVID-19 vaccines.


Subject(s)
Biotechnology/methods , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , RNA, Messenger/administration & dosage , SARS-CoV-2/immunology , Vaccines, Synthetic/administration & dosage , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , Humans , RNA, Messenger/chemistry
9.
Cell ; 184(21): 5293-5296, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1433035

ABSTRACT

The highly effective and safe mRNA-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines draw on decades of painstaking research to overcome the many hurdles for delivering, expressing, and avoiding toxicity of therapeutic mRNA. Cell editor Nicole Neuman talked with Dr. Katalin Karikó and Dr. Drew Weissman, recipients of the 2021 Lasker∼DeBakey Clinical Medical Research Award, to learn more about their quest to develop mRNA-based therapeutics, which led them to the crucial discovery that modification of mRNA could prevent toxicity and increase expression. This conversation has been adapted for print below, with editing for clarity, accuracy, and length.


Subject(s)
Biotechnology/methods , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , RNA, Messenger/administration & dosage , SARS-CoV-2/immunology , Vaccines, Synthetic/administration & dosage , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , Drug Discovery , Humans , Interviews as Topic , RNA, Messenger/chemistry
10.
Sci Signal ; 14(689)2021 06 29.
Article in English | MEDLINE | ID: covidwho-1406596

ABSTRACT

Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2'-O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn2+, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2'-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2'-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.


Subject(s)
COVID-19/virology , RNA Caps/metabolism , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Humans , Manganese/metabolism , Methylation , Methyltransferases/chemistry , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Nucleic Acid Conformation , RNA Caps/chemistry , RNA Caps/genetics , RNA Stability , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/chemistry , RNA, Viral/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolism , SARS-CoV-2/genetics , Signal Transduction , Substrate Specificity , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
11.
Nucleic Acids Res ; 49(18): 10604-10617, 2021 10 11.
Article in English | MEDLINE | ID: covidwho-1406489

ABSTRACT

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term 'superfolder' mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.


Subject(s)
Algorithms , RNA, Double-Stranded/chemistry , RNA, Messenger/chemistry , RNA, Viral/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Base Pairing , Base Sequence , COVID-19/prevention & control , Humans , Hydrolysis , RNA Stability , RNA, Double-Stranded/genetics , RNA, Double-Stranded/immunology , RNA, Messenger/genetics , RNA, Messenger/immunology , RNA, Viral/genetics , RNA, Viral/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Thermodynamics
12.
Cell ; 184(1): 184-193.e10, 2021 01 07.
Article in English | MEDLINE | ID: covidwho-1385213

ABSTRACT

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/chemistry , Cryoelectron Microscopy , RNA, Messenger/chemistry , RNA, Viral/chemistry , SARS-CoV-2/chemistry , Viral Replicase Complex Proteins/chemistry , Amino Acid Sequence , Coronavirus/chemistry , Coronavirus/classification , Coronavirus/enzymology , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Methyltransferases/metabolism , Models, Molecular , RNA Helicases/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , SARS-CoV-2/enzymology , Sequence Alignment , Transcription, Genetic , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Virus Replication
13.
Sci Rep ; 11(1): 13533, 2021 06 29.
Article in English | MEDLINE | ID: covidwho-1387483

ABSTRACT

The host receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), is highly expressed in small intestine. Our aim was to study colonic ACE2 expression in Crohn's disease (CD) and non-inflammatory bowel disease (non-IBD) controls. We hypothesized that the colonic expression levels of ACE2 impacts CD course. We examined the expression of colonic ACE2 in 67 adult CD and 14 NIBD control patients using RNA-seq and quantitative (q) RT-PCR. We validated ACE2 protein expression and localization in formalin-fixed, paraffin-embedded matched colon and ileal tissues using immunohistochemistry. The impact of increased ACE2 expression in CD for the risk of surgery was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator. To provide critical support for the generality of our findings, we analyzed previously published RNA-seq data from two large independent cohorts of CD patients. Colonic ACE2 expression was significantly higher in a subset of adult CD patients which was defined as the ACE2-high CD subset. IHC in a sampling of ACE2-high CD patients confirmed high ACE2 protein expression in the colon and ileum compared to ACE2-low CD and NIBD patients. Notably, we found that ACE2-high CD patients are significantly more likely to undergo surgery within 5 years of CD diagnosis, and a Cox regression analysis found that high ACE2 levels is an independent risk factor for surgery (OR 2.17; 95% CI, 1.10-4.26; p = 0.025). Increased intestinal expression of ACE2 is associated with deteriorated clinical outcomes in CD patients. These data point to the need for molecular stratification that can impact CD disease-related outcomes.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Crohn Disease/pathology , Adolescent , Adult , Angiotensin-Converting Enzyme 2/genetics , Crohn Disease/metabolism , Crohn Disease/surgery , Female , Humans , Ileum/metabolism , Ileum/pathology , Immunohistochemistry , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/pathology , Male , Prognosis , Proportional Hazards Models , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Risk Factors , Sequence Analysis, RNA , Young Adult
14.
J Am Chem Soc ; 143(14): 5413-5424, 2021 04 14.
Article in English | MEDLINE | ID: covidwho-1387160

ABSTRACT

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.


Subject(s)
Fluorescence , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Molecular Imaging , RNA, Messenger/analysis , RNA, Messenger/metabolism , COVID-19/drug therapy , Cell Line, Tumor , Cytosine/analogs & derivatives , Cytosine/analysis , Cytosine/chemical synthesis , Cytosine/chemistry , Fluorescent Dyes/chemical synthesis , Green Fluorescent Proteins/metabolism , Histones/metabolism , Humans , Molecular Structure , RNA, Messenger/chemistry , RNA, Messenger/therapeutic use , Spectrometry, Fluorescence
15.
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
16.
Adv Mater ; 33(34): e2101707, 2021 Aug.
Article in English | MEDLINE | ID: covidwho-1316189

ABSTRACT

The transfer of foreign synthetic messenger RNA (mRNA) into cells is essential for mRNA-based protein-replacement therapies. Prophylactic mRNA COVID-19 vaccines commonly utilize nanotechnology to deliver mRNA encoding SARS-CoV-2 vaccine antigens, thereby triggering the body's immune response and preventing infections. In this study, a new combinatorial library of symmetric lipid-like compounds is constructed, and among which a lead compound is selected to prepare lipid-like nanoassemblies (LLNs) for intracellular delivery of mRNA. After multiround optimization, the mRNA formulated into core-shell-structured LLNs exhibits more than three orders of magnitude higher resistance to serum than the unprotected mRNA, and leads to sustained and high-level protein expression in mammalian cells. A single intravenous injection of LLNs into mice achieves over 95% mRNA translation in the spleen, without causing significant hematological and histological changes. Delivery of in-vitro-transcribed mRNA that encodes high-affinity truncated ACE2 variants (tACE2v mRNA) through LLNs induces elevated expression and secretion of tACE2v decoys, which is able to effectively block the binding of the receptor-binding domain of the SARS-CoV-2 to the human ACE2 receptor. The robust neutralization activity in vitro suggests that intracellular delivery of mRNA encoding ACE2 receptor mimics via LLNs may represent a potential intervention strategy for COVID-19.


Subject(s)
COVID-19 Vaccines/genetics , Galactosidases/chemistry , Nanoparticles/chemistry , Phosphatidylethanolamines/chemistry , RNA, Messenger/chemistry , SARS-CoV-2/genetics , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/prevention & control , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/metabolism , Cell Membrane Permeability , Cell Survival/drug effects , Female , Galactosidases/metabolism , Gene Expression Regulation , Gene Transfer Techniques , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Phosphatidylethanolamines/metabolism , Protein Binding , RNA, Messenger/genetics
17.
J Immunother Cancer ; 9(6)2021 06.
Article in English | MEDLINE | ID: covidwho-1266401

ABSTRACT

SARS-CoV-2 infection and the resulting COVID-19 have afflicted millions of people in an ongoing worldwide pandemic. Safe and effective vaccination is needed urgently to protect not only the general population but also vulnerable subjects such as patients with cancer. Currently approved mRNA-based SARS-CoV-2 vaccines seem suitable for patients with cancer based on their mode of action, efficacy, and favorable safety profile reported in the general population. Here, we provide an overview of mRNA-based vaccines including their safety and efficacy. Extrapolating from insights gained from a different preventable viral infection, we review existing data on immunity against influenza A and B vaccines in patients with cancer. Finally, we discuss COVID-19 vaccination in light of the challenges specific to patients with cancer, such as factors that may hinder protective SARS-CoV-2 immune responses in the context of compromised immunity and the use of immune-suppressive or immune-modulating drugs.


Subject(s)
COVID-19 Vaccines , Neoplasms/therapy , RNA, Messenger , SARS-CoV-2/immunology , Viral Vaccines , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19 Vaccines/genetics , COVID-19 Vaccines/therapeutic use , Drug Stability , Humans , Influenza, Human/epidemiology , Influenza, Human/immunology , Influenza, Human/prevention & control , Neoplasms/epidemiology , Neoplasms/immunology , Pandemics , RNA Stability/physiology , RNA, Messenger/administration & dosage , RNA, Messenger/adverse effects , RNA, Messenger/chemistry , RNA, Messenger/genetics , SARS-CoV-2/genetics , Vaccination/methods , Viral Vaccines/adverse effects , Viral Vaccines/chemistry , Viral Vaccines/genetics
18.
Science ; 372(6548): 1306-1313, 2021 06 18.
Article in English | MEDLINE | ID: covidwho-1228853

ABSTRACT

Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.


Subject(s)
Frameshifting, Ribosomal , RNA, Viral/genetics , Ribosomes/ultrastructure , SARS-CoV-2/genetics , Viral Proteins/biosynthesis , Animals , Antiviral Agents/pharmacology , Codon, Terminator , Coronavirus RNA-Dependent RNA Polymerase/biosynthesis , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Cryoelectron Microscopy , Fluoroquinolones/pharmacology , Frameshifting, Ribosomal/drug effects , Genome, Viral , Humans , Image Processing, Computer-Assisted , Models, Molecular , Nucleic Acid Conformation , Open Reading Frames , Protein Folding , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Ribosomal, 18S/chemistry , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 18S/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Viral Proteins/chemistry , Viral Proteins/genetics , Virus Replication/drug effects
19.
Proc Natl Acad Sci U S A ; 118(21)2021 05 25.
Article in English | MEDLINE | ID: covidwho-1223143

ABSTRACT

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.


Subject(s)
RNA Caps/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , SARS-CoV-2/chemistry , Crystallography , Methylation , Methyltransferases/chemistry , Methyltransferases/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , RNA Cap Analogs/chemistry , RNA Cap Analogs/metabolism , RNA Caps/chemistry , RNA, Messenger/chemistry , RNA, Viral/chemistry , S-Adenosylhomocysteine/chemistry , S-Adenosylhomocysteine/metabolism , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Synchrotrons , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism
20.
J Am Chem Soc ; 143(14): 5413-5424, 2021 04 14.
Article in English | MEDLINE | ID: covidwho-1164792

ABSTRACT

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.


Subject(s)
Fluorescence , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Molecular Imaging , RNA, Messenger/analysis , RNA, Messenger/metabolism , COVID-19/drug therapy , Cell Line, Tumor , Cytosine/analogs & derivatives , Cytosine/analysis , Cytosine/chemical synthesis , Cytosine/chemistry , Fluorescent Dyes/chemical synthesis , Green Fluorescent Proteins/metabolism , Histones/metabolism , Humans , Molecular Structure , RNA, Messenger/chemistry , RNA, Messenger/therapeutic use , Spectrometry, Fluorescence
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