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1.
Biomater Sci ; 10(11): 2940-2952, 2022 May 31.
Article in English | MEDLINE | ID: covidwho-1815640

ABSTRACT

Ionizable cationic lipids play a critical role in developing new gene therapies for various biomedical applications, including COVID-19 vaccines. However, it remains unclear whether the formulation of lipid nanoparticles (LNPs) using DLin-MC3-DMA, an optimized ionizable lipid clinically used for small interfering RNA (siRNA) therapy, also facilitates high liver-selective transfection of other gene therapies such as plasmid DNA (pDNA). Here we report the first investigation into pDNA transfection efficiency in different mouse organs after intramuscular and intravenous administration of lipid nanoparticles (LNPs) where DLin-MC3-DMA, DLin-KC2-DMA or DODAP are used as the ionizable cationic lipid component of the LNP. We discovered that these three benchmark lipids previously developed for siRNA delivery followed an unexpected characteristic rank order in gene expression efficiency when utilized for pDNA. In particular, DLin-KC2-DMA facilitated higher in vivo pDNA transfection than DLin-MC3-DMA and DODAP, possibly due to its head group pKa and lipid tail structure. Interestingly, LNPs formulated with either DLin-KC2-DMA or DLin-MC3-DMA exhibited significantly higher in vivo protein production in the spleen than in the liver. This work sheds light on the importance of the choice of ionizable cationic lipid and nucleic acid cargo for organ-selective gene expression. The study also provides a new design principle towards the formulation of more effective LNPs for biomedical applications of pDNA, such as gene editing, vaccines and immunotherapies.


Subject(s)
COVID-19 , Nanoparticles , Animals , COVID-19 Vaccines , Cations/chemistry , DNA/genetics , Gene Expression , Humans , Lipids/chemistry , Liposomes , Mice , Nanoparticles/chemistry , Plasmids/genetics , RNA, Small Interfering/chemistry
2.
Comput Biol Chem ; 98: 107687, 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1814276

ABSTRACT

SARS-CoV-2 is a highly transmissible and pathogenic coronavirus that first emerged in late 2019 and has since triggered a pandemic of acute respiratory disease named COVID-19 which poses a significant threat to all public health institutions in the absence of specific antiviral treatment. Since the outbreak began in March 2020, India has reported 4.77 lakh Coronavirus deaths, according to the World Health Organization (WHO). The innate RNA interference (RNAi) pathway, on the other hand, allows for the development of nucleic acid-based antiviral drugs in which complementary small interfering RNAs (siRNAs) mediate the post-transcriptional gene silencing (PTGS) of target mRNA. Therefore, in this current study, the potential of RNAi was harnessed to construct siRNA molecules that target the consensus regions of specific structural proteins associated genes of SARS-CoV-2, such as the envelope protein gene (E), membrane protein gene (M), nucleocapsid phosphoprotein gene (N), and surface glycoprotein gene (S) which are important for the viral pathogenesis. Conserved sequences of 811 SARS-CoV-2 strains from around India were collected to design 21 nucleotides long siRNA duplex based on various computational algorithms and parameters targeting E, M, N and S genes. The proposed siRNA molecules possessed sufficient nucleotide-based and other features for effective gene silencing and BLAST results revealed that siRNAs' targets have no significant matches across the whole human genome. Hence, siRNAs were found to have no off-target effects on the genome, ruling out the possibility of off-target silencing. Finally, out of 157 computationally identified siRNAs, only 4 effective siRNA molecules were selected for each target gene which is proposed to exert the best action based on GC content, free energy of folding, free energy of binding, melting temperature, heat capacity and molecular docking analysis with Human AGO2 protein. Our engineered siRNA candidates could be used as a genome-level therapeutic treatment against various sequenced SARS-CoV-2 strains in India. However, future applications will necessitate additional validations in vitro and in vivo animal models.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antiviral Agents/therapeutic use , COVID-19/genetics , Molecular Docking Simulation , RNA Interference , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , SARS-CoV-2/genetics
3.
Int J Mol Sci ; 23(5)2022 Feb 22.
Article in English | MEDLINE | ID: covidwho-1699203

ABSTRACT

Since December 2019, a pandemic of COVID-19 disease, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread across the globe. At present, the Food and Drug Administration (FDA) has issued emergency approval for the use of some antiviral drugs. However, these drugs still have limitations in the specific treatment of COVID-19, and as such, new treatment strategies urgently need to be developed. RNA-interference-based gene therapy provides a tractable target for antiviral treatment. Ensuring cell-specific targeted delivery is important to the success of gene therapy. The use of nanoparticles (NPs) as carriers for the delivery of small interfering RNA (siRNAs) to specific tissues or organs of the human body could play a crucial role in the specific therapy of severe respiratory infections, such as COVID-19. In this review, we describe a variety of novel nanocarriers, such as lipid NPs, star polymer NPs, and glycogen NPs, and summarize the pre-clinical/clinical progress of these nanoparticle platforms in siRNA delivery. We also discuss the application of various NP-capsulated siRNA as therapeutics for SARS-CoV-2 infection, the challenges with targeting these therapeutics to local delivery in the lung, and various inhalation devices used for therapeutic administration. We also discuss currently available animal models that are used for preclinical assessment of RNA-interference-based gene therapy. Advances in this field have the potential for antiviral treatments of COVID-19 disease and could be adapted to treat a range of respiratory diseases.


Subject(s)
COVID-19/therapy , Drug Delivery Systems/methods , Nanoparticles/administration & dosage , RNA, Small Interfering/administration & dosage , RNAi Therapeutics/methods , Animals , COVID-19/epidemiology , COVID-19/virology , Humans , Models, Genetic , Nanoparticles/chemistry , Pandemics/prevention & control , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , SARS-CoV-2/physiology
4.
Chem Pharm Bull (Tokyo) ; 69(12): 1141-1159, 2021.
Article in English | MEDLINE | ID: covidwho-1546823

ABSTRACT

Considerable efforts have been made on the development of lipid nanoparticles (LNPs) for delivering of nucleic acids in LNP-based medicines, including a first-ever short interfering RNA (siRNA) medicine, Onpattro, and the mRNA vaccines against the coronavirus disease 2019 (COVID-19), which have been approved and are currently in use worldwide. The successful rational design of ionizable cationic lipids was a major breakthrough that dramatically increased delivery efficiency in this field. The LNPs would be expected to be useful as a platform technology for the delivery of various therapeutic modalities for genome editing and even for undiscovered therapeutic mechanisms. In this review, the current progress of my research, including the molecular design of pH-sensitive cationic lipids, their applications for various tissues and cell types, and for delivering various macromolecules, including siRNA, antisense oligonucleotide, mRNA, and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system will be described. Mechanistic studies regarding relationships between the physicochemical properties of LNPs, drug delivery, and biosafety are also summarized. Furthermore, current issues that need to be addressed for next generation drug delivery systems are discussed.


Subject(s)
Drug Carriers/chemistry , Lipids/chemistry , Liposomes/chemistry , Nanoparticles/chemistry , COVID-19/immunology , COVID-19/prevention & control , COVID-19/virology , Cations/chemistry , Hydrogen-Ion Concentration , RNA, Guide/chemistry , RNA, Guide/metabolism , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , SARS-CoV-2/isolation & purification , /metabolism
5.
Chem Biol Drug Des ; 99(2): 233-246, 2022 02.
Article in English | MEDLINE | ID: covidwho-1488186

ABSTRACT

Coronavirus (SARS-CoV-2) as a global pandemic has attracted the attention of many scientific centers to find the right treatment. We expressed and purified the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein, and specific RBD aptamers were designed using SELEX method. RNAi targeting nucleocapsid phosphoprotein was synthesized and human lung cells were inoculated with aptamer-functionalized lipid nanoparticles (LNPs) containing RNAi. The results demonstrated that RBD aptamer having KD values of 0.290 nm possessed good affinity. Based on molecular docking and efficacy prediction analysis, siRNA molecule was showed the best action. LNPs were appropriately functionalized by aptamer and contained RNAi molecules. Antiviral assay using q-PCR and ELISA demonstrated that LNP functionalized with 35 µm Apt and containing 30 nm RNAi/ml of cell culture had the best antiviral activity compared to other concentrations. Applied aptamer in the nanocarrier has two important functions. First, it can deliver the drug (RNAi) to the surface of epithelial cells. Second, by binding to the SARS-CoV-2 spike protein, it inhibits the virus entrance into cells. Our data reveal an interaction between the aptamer and the virus, and RNAi targeted the virus RNA. CT scan and the clinical laboratory tests in a clinical case study, a 36-year old man who presented with severe SARS-CoV-2, demonstrated that inhalation of 10 mg Apt-LNPs-RNAi nebulized/day for six days resulted in an improvement in consolidation and ground-glass opacity in lungs on the sixth day of treatment. Our findings suggest the treatment of SARS-CoV-2 infection through inhalation of Aptamer-LNPs-RNAi.


Subject(s)
Antiviral Agents/administration & dosage , Aptamers, Nucleotide/chemistry , COVID-19/drug therapy , Liposomes/chemistry , Nanoparticles/chemistry , RNA, Small Interfering/administration & dosage , Spike Glycoprotein, Coronavirus/metabolism , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Administration, Inhalation , Adult , Alanine/analogs & derivatives , Alanine/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , COVID-19/pathology , COVID-19/virology , Cell Line , Humans , Lung/diagnostic imaging , Lung/pathology , Male , Protein Domains/genetics , RNA Interference , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , RNA, Small Interfering/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , SELEX Aptamer Technique , Severity of Illness Index , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Viral Load/drug effects
6.
Infect Genet Evol ; 93: 104951, 2021 09.
Article in English | MEDLINE | ID: covidwho-1253387

ABSTRACT

The devastating outbreak of COVID-19 has spread all over the world and has become a global health concern. There is no specific therapeutics to encounter the COVID-19. Small interfering RNA (siRNA)-based therapy is an efficient strategy to control human viral infections employing post-transcriptional gene silencing (PTGS) through neutralizing target complementary mRNA. RNA-dependent RNA polymerase (RdRp) encoded by the viral RdRp gene as a part of the replication-transcription complex can be adopted as an acceptable target for controlling SARS-CoV-2 mediated infection. Therefore, in the current study, accessible siRNA designing tools, including significant algorithms and parameters, were rationally used to design the candidate siRNAs against SARS-COV-2 encoded RdRp. The designed siRNA molecules possessed adequate nucleotide-based and other features for potent gene silencing. The targets of the designed siRNAs revealed no significant matches within the whole human genome, ruling out any possibilities for off-target silencing by the siRNAs. Characterization with different potential parameters of efficacy allowed selecting the finest siRNA among all the designed siRNA molecules. Further, validation assessment and target site accessibility prediction also rationalized the suitability of this siRNA molecule. Molecular docking study between the selected siRNA molecule and component of RNA interference (RNAi) pathway gave an excellent outcome. Molecular dynamics of two complexes: siRNA and argonaute complex, guide RNA, and target protein complex, have shown structural stability of these proteins. Therefore, the designed siRNA molecule might act as an effective therapeutic agent against the SARS-CoV-2 at the genome level and can prevent further outbreaks of COVID-19 in humans.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/genetics , RNA, Small Interfering/genetics , SARS-CoV-2/genetics , Argonaute Proteins/chemistry , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Base Composition , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Gene Silencing , Genome, Human , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/chemistry , Sequence Alignment
7.
Comput Biol Chem ; 92: 107486, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1226281

ABSTRACT

SARS-CoV-2 is a single-stranded RNA (+) virus first identified in China and then became an ongoing global outbreak. In most cases, it is fatal in humans due to respiratory malfunction. Extensive researches are going to find an effective therapeutic technique for the treatment of SARS-CoV-2 infected individuals. In this study, we attempted to design a siRNA molecule to silence the most suitable nucleocapsid(N) gene of SARS-CoV-2, which play a major role during viral pathogenesis, replication, encapsidation and RNA packaging. At first, 270 complete N gene sequences of different strains in Bangladesh of these viruses were retrieved from the NCBI database. Different computational methods were used to design siRNA molecules. A siRNA molecule was built against these strains using the SiDirect 2.0 server. Using Mfold and the OligoCalc server, the siRNA molecule was tested for its secondary structure and GC material. The Clustal Omega tool was employed to evaluate any off-target harmony of the planned siRNA molecule. Herein, we proposed a duplex siRNA molecule that does not fit any off-target sequences for the gene silencing of SARS-CoV-2. To treat SARS-CoV-2 infections, currently, any effective therapy is not available. Our engineered siRNA molecule could give an alternative therapeutic approach against various sequenced SARS-CoV-2 strains in Bangladesh.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/metabolism , RNA Interference , RNA, Small Interfering/chemistry , RNA, Small Interfering/pharmacology , SARS-CoV-2/genetics , Bangladesh/epidemiology , COVID-19/epidemiology , Computer Simulation , Coronavirus Nucleocapsid Proteins/genetics , Gene Expression Regulation, Viral , Humans , Models, Chemical
8.
Proc Natl Acad Sci U S A ; 118(19)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1214021

ABSTRACT

To realize RNA interference (RNAi) therapeutics, it is necessary to deliver therapeutic RNAs (such as small interfering RNA or siRNA) into cell cytoplasm. A major challenge of RNAi therapeutics is the endosomal entrapment of the delivered siRNA. In this study, we developed a family of delivery vehicles called Janus base nanopieces (NPs). They are rod-shaped nanoparticles formed by bundles of Janus base nanotubes (JBNTs) with RNA cargoes incorporated inside via charge interactions. JBNTs are formed by noncovalent interactions of small molecules consisting of a base component mimicking DNA bases and an amino acid side chain. NPs presented many advantages over conventional delivery materials. NPs efficiently entered cells via macropinocytosis similar to lipid nanoparticles while presenting much better endosomal escape ability than lipid nanoparticles; NPs escaped from endosomes via a "proton sponge" effect similar to cationic polymers while presenting significant lower cytotoxicity compared to polymers and lipids due to their noncovalent structures and DNA-mimicking chemistry. In a proof-of-concept experiment, we have shown that NPs are promising candidates for antiviral delivery applications, which may be used for conditions such as COVID-19 in the future.


Subject(s)
DNA/chemistry , Drug Delivery Systems , Endosomes/metabolism , Nanostructures/administration & dosage , Amino Acids/chemistry , Cell Survival , Endocytosis , Humans , Nanostructures/chemistry , Nanotubes, Peptide/chemistry , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , RNAi Therapeutics
9.
Genomics ; 113(1 Pt 1): 331-343, 2021 01.
Article in English | MEDLINE | ID: covidwho-972544

ABSTRACT

An outbreak, caused by an RNA virus, SARS-CoV-2 named COVID-19 has become pandemic with a magnitude which is daunting to all public health institutions in the absence of specific antiviral treatment. Surface glycoprotein and nucleocapsid phosphoprotein are two important proteins of this virus facilitating its entry into host cell and genome replication. Small interfering RNA (siRNA) is a prospective tool of the RNA interference (RNAi) pathway for the control of human viral infections by suppressing viral gene expression through hybridization and neutralization of target complementary mRNA. So, in this study, the power of RNA interference technology was harnessed to develop siRNA molecules against specific target genes namely, nucleocapsid phosphoprotein gene and surface glycoprotein gene. Conserved sequence from 139 SARS-CoV-2 strains from around the globe was collected to construct 78 siRNA that can inactivate nucleocapsid phosphoprotein and surface glycoprotein genes. Finally, based on GC content, free energy of folding, free energy of binding, melting temperature, efficacy prediction and molecular docking analysis, 8 siRNA molecules were selected which are proposed to exert the best action. These predicted siRNAs should effectively silence the genes of SARS-CoV-2 during siRNA mediated treatment assisting in the response against SARS-CoV-2.


Subject(s)
COVID-19/therapy , Computational Chemistry , Coronavirus Nucleocapsid Proteins/genetics , Drug Design , Genetic Therapy/methods , Molecular Docking Simulation , RNA Interference , RNA, Messenger/antagonists & inhibitors , RNA, Small Interfering/chemistry , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Argonaute Proteins/chemistry , Argonaute Proteins/genetics , Base Composition , COVID-19/drug therapy , COVID-19/virology , Evolution, Molecular , Gene Expression Regulation, Viral/drug effects , Humans , Pandemics , Phosphoproteins/genetics , Phylogeny , RNA Folding , RNA, Guide/chemistry , RNA, Guide/genetics , RNA, Messenger/genetics , RNA, Small Interfering/pharmacology , RNA, Small Interfering/therapeutic use , RNA, Viral/genetics , SARS-CoV-2/drug effects , Sequence Alignment , Thermodynamics
10.
Nanoscale ; 12(47): 23959-23966, 2020 Dec 21.
Article in English | MEDLINE | ID: covidwho-947558

ABSTRACT

Lipid nanoparticle (LNP) formulations of nucleic acid are leading vaccine candidates for COVID-19, and enabled the first approved RNAi therapeutic, Onpattro. LNPs are composed of ionizable cationic lipids, phosphatidylcholine, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced using rapid-mixing techniques. These procedures involve dissolution of the lipid components in an organic phase and the nucleic acid in an acidic aqueous buffer (pH 4). These solutions are then combined using a continuous mixing device such as a T-mixer or microfluidic device. In this mixing step, particle formation and nucleic acid entrapment occur. Previous work from our group has shown that, in the absence of nucleic acid, the particles formed at pH 4 are vesicular in structure, a portion of these particles are converted to electron-dense structures in the presence of nucleic acid, and the proportion of electron-dense structures increases with nucleic acid content. What remained unclear from previous work was the mechanism by which vesicles form electron-dense structures. In this study, we use cryogenic transmission electron microscopy and dynamic light scattering to show that efficient siRNA entrapment occurs in the absence of ethanol (contrary to the established paradigm), and suggest that nucleic acid entrapment occurs through inversion of preformed vesicles. We also leverage this phenomenon to show that specialized mixers are not required for siRNA entrapment, and that preformed particles at pH 4 can be used for in vitro transfection.


Subject(s)
COVID-19 , Lab-On-A-Chip Devices , Lipids , Nanoparticles , RNA, Small Interfering , SARS-CoV-2 , Animals , Cell Line , Hydrogen-Ion Concentration , Lipids/chemistry , Lipids/pharmacology , Mice , Nanoparticles/chemistry , Nanoparticles/therapeutic use , RNA, Small Interfering/chemistry , RNA, Small Interfering/pharmacology
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