Biogenically synthesized green silver nanoparticles exhibit antimalarial activity.

The suboptimal efficacies of existing anti-malarial drugs attributed to the emergence of drug resistance dampen the clinical outcomes. Hence, there is a need for developing novel drug and drug targets. Recently silver nanoparticles (AgNPs) constructed with the leaf extracts of Euphorbia cotinifolia were shown to possess antimalarial activity. Therefore, the synthesized AgNPs from Euphorbia cotinifolia (EcAgNPs) were tested for their parasite clearance activity. We determined the antimalarial activity in the asexual blood stage infection of 3D7 (laboratory strain) P. falciparum. EcAgNPs demonstrated the significant inhibition of parasite growth (EC50 of 0.75 µg/ml) in the routine in vitro culture of P. falciparum. The synthesized silver nanoparticles were seen to induce apoptosis in P. falciparum through increased reactive oxygen species (ROS) ROS production and activated programmed cell death pathways characterized by the caspase-3 and calpain activity. Also, altered transcriptional regulation of Bax/Bcl-2 ratio indicated the enhanced apoptosis. Moreover, inhibited expression of PfLPL-1 by EcAgNPs is suggestive of the dysregulated host fatty acid flux via parasite lipid storage. Overall, our findings suggest that EcAgNPs are a non-toxic and targeted antimalarial treatment, and could be a promising therapeutic approach for clearing malaria infection.

Base-modified factor VIII mRNA delivery with galactosylated lipid nanoparticles as a protein replacement therapy for haemophilia A.

The bleeding disorder hemophilia A (HemA) requires systemic functional factor VIII protein infusions on prophylactic schedules. Recently, chemically modified mRNAs have emerged as promising protein replacement therapies to reduce repeated infusions and improve safety profiles. However, the influence of base modifications on mRNA translation kinetics to specific cell types remains unexplored. In this study, towards developing mRNA therapeutics for haemophilia A, we synthesized chemically modified mRNAs with commercially available base modifications of adenine, guanine, uridine, and cytidine, and evaluated in vitro transcription yield and translation kinetics in hepatic cell lines using reporter eGFP mRNA. Our findings demonstrated that mRNA with N1-methyl pseudouridine (m1Ψ) showed a 5-12-fold increase in translation efficiency in both hepatic and endothelial cell lines. As a proof of concept for developing mRNA therapy for HemA, where FVIII is deficient, we developed a m1Ψ modified functional FVIII mRNA with our liver-targeting lipid nanoparticle (Gal-LNP) system. We evaluated its delivery efficiencies in both hepatic cell lines and the HemA mouse model. The m1Ψ-FVIII mRNA showed high therapeutic efficacy up to 15 days in vivo in the HemA mouse model. Gal-LNPs were found to be safe for systemic administration. Our study reveals that incorporating m1Ψ base modifications on mRNAs could improve therapeutic efficacy in liver- and endothelial-based therapeutics. Optimized mRNA synthesis for superior expression kinetics in hepatic cells and its delivery with liver-targeted nanoparticles may emerge as protein replacement therapies for monogenic liver disorders.

Lipid-nanoparticle-enabled nucleic acid therapeutics for liver disorders.

Inherited genetic disorders of the liver pose a significant public health burden. Liver transplantation is often limited by the availability of donor livers and the exorbitant costs of immunosuppressive therapy. To overcome these limitations, nucleic acid therapy provides a hopeful alternative that enables gene repair, gene supplementation, and gene silencing with suitable vectors. Though viral vectors are the most efficient and preferred for gene therapy, pre-existing immunity debilitating immune responses limit their use. As a potential alternative, lipid nanoparticle-mediated vectors are being explored to deliver multiple nucleic acid forms, including pDNA, mRNA, siRNA, and proteins. Herein, we discuss the broader applications of lipid nanoparticles, from protein replacement therapy to restoring the disease mechanism through nucleic acid delivery and gene editing, as well as multiple preclinical and clinical studies as a potential alternative to liver transplantation.

Role of prior immunity in binding to spike of "future" Omicron subvariants.

BACKGROUND: Throughout the COVID-19 pandemic, virus evolution and large-scale vaccination programs have caused multiple exposures to SARS CoV-2 spike protein, resulting in complex antibody profiles. The binding of these to spike protein of "future" variants in the context of such heterogeneous exposure has not been studied. METHODS: We tested archival sera (Delta and Omicron period) stratified by anti-spike antibody (including IgG) levels for reactivity to Omicron-subvariants(BA.1, BA.2,BA.2.12.1, BA.2.75, BA.4/5 and BF.7) spike protein. Assessed antigenic distance between groups using Antigenic Cartography and performed hierarchical clustering of antibody data in a Euclidean distance framework. RESULTS: Antibody (including IgG) antibody reactivity to Wild-type (CLIA) and subvariants (ELISA) spike protein were similar between periods (p > 0.05). Both 'High S' and 'Low S' of Delta and Omicron periods were closely related to "future" subvariants by Antigenic Cartography. Sera from different S groups clustered together with 'Low S' interspersed between 'High S' on hierarchical clustering, suggesting common binding sites. Further, anti-spike antibodies (including IgG) to Wild-type (S1/S2 and Trimeric S) clustered with Omicron-subvariant binding antibodies. CONCLUSIONS: Hybrid immunity caused by cumulative virus exposure in Delta or Omicron periods resulted in equivalent binding to "future" variants, which might be due to binding to conserved regions of spike protein of future variants. A prominent finding is that the 'Low S' antibody demonstrates similar binding.

Precise correction of a spectrum of ß-thalassemia mutations in coding and non-coding regions by base editors.

ß-thalassemia/HbE results from mutations in the ß-globin locus that impede the production of functional adult hemoglobin. Base editors (BEs) could facilitate the correction of the point mutations with minimal or no indel creation, but its efficiency and bystander editing for the correction of ß-thalassemia mutations in coding and non-coding regions remains unexplored. Here, we screened BE variants in HUDEP-2 cells for their ability to correct a spectrum of ß-thalassemia mutations that were integrated into the genome as fragments of HBB. The identified targets were introduced into their endogenous genomic location using BEs and Cas9/homology-directed repair (HDR) to create cellular models with ß-thalassemia/HbE. These ß-thalassemia/HbE models were then used to assess the efficiency of correction in the native locus and functional ß-globin restoration. Most bystander edits produced near target sites did not interfere with adult hemoglobin expression and are not predicted to be pathogenic. Further, the effectiveness of BE was validated for the correction of the pathogenic HbE variant in severe ß0/ßE-thalassaemia patient cells. Overall, our study establishes a novel platform to screen and select optimal BE tools for therapeutic genome editing by demonstrating the precise, efficient, and scarless correction of pathogenic point mutations spanning multiple regions of HBB including the promoter, intron, and exons.

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.

Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression.

BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for ß-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.

Current approaches and potential challenges in the delivery of gene editing cargos into hematopoietic stem and progenitor cells.

Advancements in gene delivery and editing have expanded the applications of autologous hematopoietic stem and progenitor cells (HSPCs) for the treatment of monogenic and acquired diseases. The gene editing toolbox is growing, and the ability to achieve gene editing with mRNA or protein delivered intracellularly by vehicles, such as electroporation and nanoparticles, has highlighted the potential of gene editing in HSPCs. Ongoing phase I/II clinical trials with gene-edited HSPCs for ß-hemoglobinopathies provide hope for treating monogenic diseases. The development of safe and efficient gene editing reagents and their delivery into hard-to-transfect HSPCs have been critical drivers in the rapid translation of HSPC gene editing into clinical studies. This review article summarizes the available payloads and delivery vehicles for gene editing HSPCs and their potential impact on therapeutic applications.

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.

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of ß-hemoglobinopathies.

Reactivation of fetal hemoglobin (HbF) is a commonly adapted strategy to ameliorate ß-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits HbF tetramer production affecting the therapeutic benefits. Here, we evaluated deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified an 11-kb sequence, encompassing putative repressor region (PRR) to ß-globin exon-1 (ßE1), as the core deletion that ablates HbA and exhibits superior HbF production compared with HPFH or other well-established targets. PRR-ßE1-edited hematopoietic stem and progenitor cells (HSPCs) retained their genome integrity and their engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice producing HbF positive cells in vivo. Furthermore, PRR-ßE1 gene editing is feasible without ex vivo HSPC culture. Importantly, the editing induced therapeutically significant levels of HbF to reverse the phenotypes of both sickle cell disease and ß-thalassemia major. These findings imply that PRR-ßE1 gene editing of patient HSPCs could lead to improved therapeutic outcomes for ß-hemoglobinopathy gene therapy.

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.

Effect of Methylation of the Hydrophilic Domain of Tocopheryl Ammonium-Based Lipids on their Nucleic Acid Delivery Properties.

Lipid-enabled nucleic acid delivery has garnered tremendous attention in recent times. Tocopherol among the cationic lipids, 3b-[N-(N',N'-dimethylamino-ethane)carbamoyl]-cholesterol hydrochloride (DC-Chol) with a headgroup of dimethylammonium, and cholesterol as a hydrophobic moiety are found to be some of the most successful lipids and are being used in clinical trials. However, limited efficacy is a major limitation for their broader therapeutic application. In our prior studies, we demonstrated tocopherol to be a potential alternative hydrophobic moiety having additional antioxidant properties to develop efficient and safer liposomal formulations. Inspired by DC-Chol applications and taking cues from our own prior findings, herein, we report the design and synthesis of four alpha-tocopherol-based cationic derivatives with varying degrees of methylation, AC-Toc (no methylation), MC-Toc (monomethylation derivative), DC-Toc (dimethylation derivative), and TC-Toc (trimethylation derivative) and the evaluation of their gene delivery properties. The transfection studies showed that AC-Toc liposomes exhibited superior transfection compared to MC-Toc, DC-Toc, TC-Toc, and control DC-Chol, indicating that methylation in the hydrophilic moiety of Toc-lipids reduced their transfection properties. Cellular internalization studies in the presence of different endocytosis blockers revealed that all four tocopherol lipids were internalized through clathrin-mediated endocytosis, whereas control DC-Chol was found to be internalized through both macropinocytosis and clathrin-mediated endocytosis. These novel Toc-lipids exhibited higher antioxidant properties than DC-Chol by generating less reactive oxygen species, indicating lower cytotoxicity. Our present findings suggest that AC-Toc may be considered as an alternative to DC-Chol in liposomal transfections.

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.

The CCR5 Gene Edited CD34+CD90+ Hematopoietic Stem Cell Population Serves as an Optimal Graft Source for HIV Gene Therapy.

Transplantation of allogenic hematopoietic stem and progenitor cells (HSPCs) with C-C chemokine receptor type 5 (CCR5) Δ32 genotype generates HIV-1 resistant immune cells. CCR5 gene edited autologous HSPCs can be a potential alternative to hematopoietic stem cell transplantation (HSCT) from HLA-matched CCR5 null donor. However, the clinical application of gene edited autologous HSPCs is critically limited by the quality of the graft, as HIV also infects the HSPCs. In this study, by using mobilized HSPCs from healthy donors, we show that the CD34+CD90+ hematopoietic stem cells (HSCs) express 7-fold lower CD4/CCR5 HIV receptors, higher levels of SAMHD1 anti-viral restriction factor, and possess lower susceptibility to HIV infection than the CD34+CD90- hematopoietic progenitor cells. Further, the treatment with small molecule cocktail of Resveratrol, UM729 and SR1(RUS) improved the in vivo engraftment potential of CD34+CD90+ HSCs. To demonstrate that CD34+CD90+ HSC population as an ideal graft for HIV gene therapy, we sort purified CD34+CD90+ HSCs, treated with RUS and then gene edited the CCR5 with single sgRNA. On transplantation, 100,000 CD34+CD90+ HSCs were sufficient for long-term repopulation of the entire bone marrow of NBSGW mice. Importantly, the gene editing efficiency of ~90% in the infused product was maintained in vivo, facilitating the generation of CCR5 null immune cells, resistant to HIV infection. Altogether, CCR5 gene editing of CD34+CD90+ HSCs provide an ideal gene manipulation strategy for autologous HSCT based gene therapy for HIV infection.

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.

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.