SMART-lipid nanoparticles enabled mRNA vaccine elicits cross-reactive humoral responses against the omicron sub-variants.

The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has necessitated the development of broad cross-reactive vaccines. Recent findings suggest that enhanced antigen presentation could lead to cross-reactive humoral responses against the emerging variants. Toward enhancing the antigen presentation to dendritic cells (DCs), we developed a novel shikimoylated mannose receptor targeting lipid nanoparticle (SMART-LNP) system that could effectively deliver mRNAs into DCs. To improve the translation of mRNA, we developed spike domain-based trimeric S1 (TS1) mRNA with optimized codon sequence, base modification, and engineered 5' and 3' UTRs. In a mouse model, SMART-LNP-TS1 vaccine could elicit robust broad cross-reactive IgGs against Omicron sub-variants, and induced interferon-γ-producing T cells against SARS-CoV-2 virus compared with non-targeted LNP-TS1 vaccine. Further, T cells analysis revealed that SMART-LNP-TS1 vaccine induced long-lived memory T cell subsets, T helper 1 (Th1)-dominant and cytotoxic T cells immune responses against the SARS-CoV-2 virus. Importantly, SMART-LNP-TS1 vaccine produced strong Th1-predominant humoral and cellular immune responses. Overall, SMART-LNPs can be explored for precise antigenic mRNA delivery and robust immune responses. This platform technology can be explored further as a next-generation delivery system for mRNA-based immune therapies.

Correlating the differences in the receptor binding domain of SARS-CoV-2 spike variants on their interactions with human ACE2 receptor.

Spike glycoprotein of SARS-CoV-2 variants plays a critical role in infection and transmission through its interaction with human angiotensin converting enzyme 2 (hACE2) receptors. Prior findings using molecular docking and biomolecular studies reported varied findings on the difference in the interactions among the spike variants with the hACE2 receptors. Hence, it is a prerequisite to understand these interactions in a more precise manner. To this end, firstly, we performed ELISA with trimeric spike glycoproteins of SARS-CoV-2 variants including Wuhan Hu-1(Wild), Delta, C.1.2 and Omicron. Further, to study the interactions in a more specific manner by mimicking the natural infection, we developed hACE2 receptors expressing HEK-293T cell line, evaluated their binding efficiencies and competitive binding of spike variants with D614G spike pseudotyped virus. In line with the existing findings, we observed that Omicron had higher binding efficiency compared to Delta in both ELISA and Cellular models. Intriguingly, we found that cellular models could differentiate the subtle differences between the closely related C.1.2 and Delta in their binding to hACE2 receptors. Our study using the cellular model provides a precise method to evaluate the binding interactions between spike sub-lineages to hACE2 receptors.

Omicron infection increases IgG binding to spike protein of predecessor variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in India in 2020-2022 was driven predominantly by Wild (Wuhan-Hu-1 and D614G), Delta, and Omicron variants. The aim of this study was to examine the effect of infections on the humoral immune response and cross-reactivity to spike proteins of Wuhan-Hu-1, Delta, C.1.2., and Omicron. Residual archival sera (N = 81) received between January 2020 and March 2022 were included. Infection status was inferred by a positive SARS-CoV-2 RT-PCR and/or serology (anti-N and anti-S antibodies) and sequencing of contemporaneous samples (N = 18) to infer lineage. We estimated the levels and cross-reactivity of infection-induced sera including Wild, Delta, Omicron as well as vaccine breakthrough infections (Delta and Omicron). We found an approximately two-fold increase in spike-specific IgG antibody binding in post-Omicron infection compared with the pre-Omicron period, whilst the change in pre- and post-Delta infections were similar. Further investigation of Omicron-specific humoral responses revealed primary Omicron infection as an inducer of cross-reactive antibodies against predecessor variants, in spite of the weaker degree of humoral response compared to Wuhan-Hu-1 and Delta infection. Intriguingly, Omicron vaccine-breakthrough infections when compared with primary infections, exhibited increased humoral responses against RBD (7.7-fold) and Trimeric S (Trimeric form of spike protein) (34.6-fold) in addition to increased binding of IgGs towards previously circulating variants (4.2 - 6.5-fold). Despite Delta breakthrough infections showing a higher level of humoral response against RBD (2.9-fold) and Trimeric S (5.7-fold) compared to primary Delta sera, a demonstrably reduced binding (36%-49%) was observed to Omicron spike protein. Omicron vaccine breakthrough infection results in increased intensity of humoral response and wider breadth of IgG binding to spike proteins of antigenically-distinct, predecessor variants.

Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells.

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic applications, including a novel class of vaccines, protein replacement therapies, and immune therapies. Among delivery vectors, lipid nanoparticles are found to be safer and more effective in delivering RNA molecules (e.g., siRNA, miRNA, mRNA) and a few products are already in clinical use. To demonstrate lipid nanoparticle-mediated mRNA delivery, we present an optimized protocol for the synthesis of functional me1Ψ-UTP modified eGFP mRNA, the preparation of cationic liposomes, the electrostatic complex formation of mRNA with cationic liposomes, and the evaluation of transfection efficiencies in mammalian cells. The results demonstrate that these modifications efficiently improved the stability of mRNA when delivered with cationic liposomes and increased the eGFP mRNA translation efficiency and stability in mammalian cells. This protocol can be used to synthesize the desired mRNA and transfect with cationic liposomes for target gene expression in mammalian cells.

Optimization of SARS-CoV-2 Pseudovirion Production in Lentivirus Backbone With a Novel Liposomal System.

Due to the fast mutating nature of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of novel therapeutics, vaccines, and evaluating the efficacies of existing one's against the mutated strains is critical for containing the virus. Pseudotyped SARS-CoV-2 viruses are proven to be instrumental in evaluating the efficiencies of therapeutics, owing to their ease in application and safety when compared to handling the live virus. However, a comprehensive protocol that includes selecting transfection reagents, validating different packaging systems for high-throughput screening of neutralizing antibodies, is still a requisite. To this end, we designed and synthesized amide linker-based cationic lipids with varying hydrophilic head groups from dimethyl (Lipo-DME) to methyl, ethylhydroxyl (Lipo-MeOH), and diethylhydroxyl (Lipo-DOH) keeping the hydrophobic tail, stearic acid, as constant. Among the liposomal formulations of these lipids, Lipo-DOH was found to be superior in delivering plasmids and demonstrated comparable transfection efficiencies with commercial standard Lipofectamine 3000. We further used Lipo-DOH for lentivirus and SARS-CoV-2 pseudovirion preparation. For comparing different lentivirus packaging systems, we optimized conditions using Addgene and BEI systems and found that the BEI lenti plasmid system was found to be efficient in making lentiviruses using Lipo-DOH. Using the optimized transfection reagent and the lentivirus system, we developed a robust protocol for the generation of SARS-CoV-2 pseudovirions and characterized their infectivity in human ACE2 expressing HEK-293T cells and neutralizing properties in IgG against spike protein of SARS-CoV-2 positive human sera from individuals recovered from COVID-19.

Skin-Permeable Nano-Lithocholic Lipidoid Efficiently Alleviates Psoriasis-like Chronic Skin Inflammations.

Long-term application of topical therapeutics for psoriasis has a plethora of side effects. Additionally, skin-permeating agents used in their formulations for deeper dermal delivery damage the skin. To address these limitations, we developed novel lithocholic acid analogues that could form lipid nanoparticles (nano-LCs) spontaneously in the aqueous milieu, permeate through the skin, penetrate the deeper dermal layers, and exert anti-inflammatory effects against psoriasis-like chronic skin inflammations. Prior findings demonstrated that lithocholic acid acts as a vitamin D receptor agonist without affecting the Ca+2 metabolism and also as an antagonist for ephrin type-A receptor 2 (EphA2). Taking cues from the previous findings, lithocholic acid derivatives with twin alkyl chains (LC6, LC8, LC10, and LC-12) were synthesized, nanoparticles (nano-LCs) were prepared, and they were evaluated for their skin permeability and anti-inflammatory properties. Among these nano-LCs, nano-LC10 demonstrated superior anti-inflammatory properties and inhibition of keratinocyte proliferation in various cell-based evaluations. Furthermore, the therapeutic efficiency of nano-LC10 was evaluated in an imiquimod-induced psoriasis-like mouse model and demonstrated comparable efficiency with the standard topical formulation, Sorvate, in reducing skin inflammations. Nano-LC10 also reduced systemic inflammation, organ toxicity, and also proinflammatory serum cytokine levels. Overall, nano-lithocholic lipidoid (nano-LC10) can be a potential novel class of therapeutics for topical application in treating psoriasis.

Influence of Hydrophobicity in the Hydrophilic Region of Cationic Lipids on Enhancing Nucleic Acid Delivery and Gene Editing.

Intracellular delivery of biomolecules using non-viral vectors critically depends on the vectors' ability to allow the escape and release of the contents from the endosomes. Prior findings demonstrated that aromatic/hydrophobic group-containing amino acids such as phenylalanine (F) and tryptophan (W) destabilize cellular membranes by forming pores in the lipid bilayer. Taking cues from these findings, we have developed four α-tocopherol-based cationic amphiphiles by varying the aromatic/hydrophobic amino acids such as glycine (G), proline (P), phenylalanine (F), and tryptophan (W) as head groups and triazole in the linker region to study their impact on endosomal escape for the enhanced transfection efficacy. The lipids tocopherol-triazole-phenylalanine (TTF) and tocopherol-triazole-tryptophan (TTW) exhibited similar potential to commercial transfecting reagents, Lipofectamine (LF) 3000 and Lipofectamine Messenger Max (LFMM), respectively, in transfecting plasmid DNA and messenger RNA in multiple cultured cell lines. The TTW liposome was also found to be effective in delivering Cas9 mRNA and demonstrated equal efficiency of gene editing AAVS1 locus compared to LFMM in CHO, Neuro-2a, and EA.HY926 cell lines. In this current investigation, it is shown that the synthesized cationic lipids with aromatic hydrophobic R group-containing amino acids are safe, economic, and actually more efficient in nucleic acid delivery and genome-editing applications. These findings can be further explored in the genome-editing approach for treating genetic disorders.

Diosgenin enhances liposome-enabled nucleic acid delivery and CRISPR/Cas9-mediated gene editing by modulating endocytic pathways.

The CRISPR/Cas9 system holds great promise in treating genetic diseases, owing to its safe and precise genome editing. However, the major challenges to implementing the technology in clinics lie in transiently limiting the expression of genome editing factors and achieving therapeutically relevant frequencies with fidelity. Recent findings revealed that non-viral vectors could be a potential alternative delivery system to overcome these limitations. In our previous research, we demonstrated that liposomal formulations with amide linker-based cationic lipids and cholesterol were found to be effective in delivering a variety of nucleic acids. In the current study, we screened steroidal sapogenins as an alternative co-lipid to cholesterol in cationic liposomal formulations and found that liposomes with diosgenin (AD, Amide lipid: Diosgenin) further improved nucleic acid delivery efficacy, in particular, delivering Cas9 pDNA and mRNA for efficient genome editing at multiple loci, including AAVS1 and HBB, when compared to amide cholesterol. Mechanistic insights into the endocytosis of lipoplexes revealed that diosgenin facilitated the lipoplexes' cholesterol-independent and clathrin-mediated endocytosis, which in turn leads to increased intracellular delivery. Our study identifies diosgenin-doped liposomes as an efficient tool to deliver CRISPR/Cas9 system.

Controlled Differentiation of Mesenchymal Stem Cells into Hyaline Cartilage in miR-140-Activated Collagen Hydrogel.

OBJECTIVE: Hypertrophic cartilage formation is a major setback in mesenchymal stem cells (MSCs)-mediated cartilage repair, and overcoming it requires optimization of differentiation. Here, we tested the miR-140 activated collagen hydrogel for the chondrogenic differentiation of MSCs and to produce hyaline cartilage. METHODS: Bone marrow MSCs isolated from 3 patients were pretreated with miR-140 and then chondrogenic differentiated. The 3-dimensional (3D) transfection potential of 5 different transfection reagents (Polyethylenimine, Lipofectamine, TransIT-X2, Amide:Cholesterol-based liposomes [AmC] and AmC pegylated with Tocofersolan [AmCTOC]) was compared and the reagent that showed higher green fluorescent protein (GFP) expression was selected. Finally, the collagen hydrogel was activated using miR-140-transfection complex and sustained delivered to MSCs during chondrogenic differentiation. After differentiation, the outcome was assessed by reverse transcription-polymerase chain reaction (RT-PCR), histology, immunohistochemistry, and compared with scrambled miRNA treated control. RESULTS: Pretreatment of MSCs with miR-140 significantly increased the expression of cartilage-specific genes (COL2A1, SOX9, and ACAN) with reduced hypertrophic chondrocyte (COL10A1) marker expression and better safranin-O staining than the control. The AmCTOC liposome showed a significant increase in 3D transfection of GFP expressing plasmid than the others. Furthermore, the knockdown of GAPDH using siRNA in HEK cells and expression of GFP mRNA in human bone marrow MSCs confirmed the 3D-transfection efficiency of AmCTOC. The sustained delivery of miR-140 using activated matrix formed a hyaline cartilage-like tissue with minimal COL10A1 expression in RT-PCR and immunohistochemistry. CONCLUSION: Our results demonstrated the therapeutic potential of miR-140-activated hydrogel for MSCs-based cartilage tissue engineering, which could also be used for endogenous stem cells-mediated cartilage repair.

Cholesterol Sequestration from Caveolae/Lipid Rafts Enhances Cationic Liposome-Mediated Nucleic Acid Delivery into Endothelial Cells.

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.