Improved transfer efficiency of supercharged 36 + GFP protein mediate nucleic acid delivery.

The potential of nucleic acid therapeutics to treat diseases by targeting specific cells has resulted in its increasing number of uses in clinical settings. However, the major challenge is to deliver bio-macromolecules into target cells and/or subcellular locations of interest ahead in the development of delivery systems. Although, supercharged residues replaced protein 36 + GFP can facilitate itself and cargoes delivery, its efficiency is still limited. Therefore, we combined our recent progress to further improve 36 + GFP based delivery efficiency. We found that the penetration efficacy of 36 + GFP protein was significantly improved by fusion with CPP-Dot1l or treatment with penetration enhancer dimethyl sulfoxide (DMSO) in vitro. After safely packaged with plasmid DNA, we found that the efficacy of in vitro and in vivo transfection mediated by 36 + GFP-Dot1l fusion protein is also significantly improved than 36 + GFP itself. Our findings illustrated that fusion with CPP-Dot1l or incubation with DMSO is an alternative way to synergically promote 36 + GFP mediated plasmid DNA delivery in vitro and in vivo.

Gene-Delivery Ability of New Hydrogenated and Partially Fluorinated Gemini bispyridinium Surfactants with Six Methylene Spacers.

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.

The evaluation of novel oral vaccines based on self-amplifying RNA lipid nanparticles (saRNA LNPs), saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum to neutralize SARS-CoV-2 variants alpha and delta.

The aim of this study was to present and evaluate novel oral vaccines, based on self-amplifying RNA lipid nanparticles (saRNA LNPs), saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum, to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) variants alpha and delta. After invitro evaluation of the oral vaccines on HEK293T/17 cells, we found that saRNA LNPs, saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum could express S-protein at both mRNA and protein levels. In the next step, BALB/c mice were orally vaccinated with saRNA LNPs, saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum at weeks 1 and 3. Importantly, a high titer of IgG and IgA was observed by all of them, sharply in week 6 (P < 0.05). In all study groups, their ratio of IgG2a/IgG1 was upper 1, indicating Th1-biased responses. Wild-type viral neutralization assay showed that the secreted antibodies in vaccinated mice and recovered COVID-19 patients could neutralize SARS-COV-2 variants alpha and delta. After oral administration of oral vaccines, biodistribution assay was done. It was found that all of them had the same biodistribution pattern. The highest concentration of S-protein was seen in the small intestine, followed by the large intestine and liver.

Reproducible and Easy Production of Mammalian Proteins by Transient Gene Expression in High Five Insect Cells.

The expression of mammalian recombinant proteins in insect cell lines using transient-plasmid-based gene expression enables the production of high-quality protein samples. Here, the procedure for virus-free transient gene expression (TGE) in High Five insect cells is described in detail. The parameters that determine the efficiency and reproducibility of the method are presented in a robust protocol for easy implementation and set-up of the method. The applicability of the TGE method in High Five cells for proteomic, structural, and functional analysis of the expressed proteins is shown.

Direct reprogramming induces vascular regeneration post muscle ischemic injury.

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail, termed 7G-modRNA, of four cardiac-reprogramming genes-Gata4 (G), Mef2c (M), Tbx5 (T), and Hand2 (H)-together with three reprogramming-helper genes-dominant-negative (DN)-TGFß, DN-Wnt8a, and acid ceramidase (AC)-to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival, and capillary density. Mechanistically, we determined that while 7G-modRNA cannot create de novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.

Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs.

Nucleoside-modified messenger RNA (mRNA)-lipid nanoparticles (LNPs) are the basis for the first two EUA (Emergency Use Authorization) COVID-19 vaccines. The use of nucleoside-modified mRNA as a pharmacological agent opens immense opportunities for therapeutic, prophylactic and diagnostic molecular interventions. In particular, mRNA-based drugs may specifically modulate immune cells, such as T lymphocytes, for immunotherapy of oncologic, infectious and other conditions. The key challenge, however, is that T cells are notoriously resistant to transfection by exogenous mRNA. Here, we report that conjugating CD4 antibody to LNPs enables specific targeting and mRNA interventions to CD4+ cells, including T cells. After systemic injection in mice, CD4-targeted radiolabeled mRNA-LNPs accumulated in spleen, providing ∼30-fold higher signal of reporter mRNA in T cells isolated from spleen as compared with non-targeted mRNA-LNPs. Intravenous injection of CD4-targeted LNPs loaded with Cre recombinase-encoding mRNA provided specific dose-dependent loxP-mediated genetic recombination, resulting in reporter gene expression in about 60% and 40% of CD4+ T cells in spleen and lymph nodes, respectively. T cell phenotyping showed uniform transfection of T cell subpopulations, with no variability in uptake of CD4-targeted mRNA-LNPs in naive, central memory, and effector cells. The specific and efficient targeting and transfection of mRNA to T cells established in this study provides a platform technology for immunotherapy of devastating conditions and HIV cure.

Safe and effective aerosolization of in vitro transcribed mRNA to the respiratory tract epithelium of horses without a transfection agent.

Vaccines and therapeutics using in vitro transcribed mRNA hold enormous potential for human and veterinary medicine. Transfection agents are widely considered to be necessary to protect mRNA and enhance transfection, but they add expense and raise concerns regarding quality control and safety. We found that such complex mRNA delivery systems can be avoided when transfecting epithelial cells by aerosolizing the mRNA into micron-sized droplets. In an equine in vivo model, we demonstrated that the translation of mRNA into a functional protein did not depend on the addition of a polyethylenimine (PEI)-derived transfection agent. We were able to safely and effectively transfect the bronchial epithelium of foals using naked mRNA (i.e., mRNA formulated in a sodium citrate buffer without a delivery vehicle). Endoscopic examination of the bronchial tree and histology of mucosal biopsies indicated no gross or microscopic adverse effects of the transfection. Our data suggest that mRNA administered by an atomization device eliminates the need for chemical transfection agents, which can reduce the cost and the safety risks of delivering mRNA to the respiratory tract of animals and humans.

High-level expression of the monomeric SARS-CoV-2 S protein RBD 320-537 in stably transfected CHO cells by the EEF1A1-based plasmid vector.

The spike (S) protein is one of the three proteins forming the coronaviruses' viral envelope. The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (RBD), which is a significant inducer of host immune response. Recombinant SARS-CoV-2 RBD is widely used as a highly specific minimal antigen for serological tests. Correct exposure of antigenic determinants has a significant impact on the accuracy of such tests-the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native S protein. Based on the previously developed p1.1 vector, containing the regulatory sequences of the Eukaryotic translation elongation factor 1 alpha gene (EEF1A1) from Chinese hamster, we created two expression constructs encoding SARS-CoV-2 RBD with C-terminal c-myc and polyhistidine tags. RBDv1 contained a native viral signal peptide, RBDv2 -human tPA signal peptide. We transfected a CHO DG44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. For the RBDv2 variant, a high-yield clonal producer cell line was obtained. We developed a simple purification scheme that consistently yielded up to 30 mg of RBD protein per liter of the simple shake flask cell culture. Purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for RBDv2 protein, the monomeric form content exceeded 90% for several series. Deglycosylation with PNGase F and mass spectrometry confirmed the presence of N-glycosylation. The antigen produced by the described technique is suitable for serological tests and subunit vaccine studies.

Hybrid Biopolymer and Lipid Nanoparticles with Improved Transfection Efficacy for mRNA.

Hybrid nanoparticles from lipidic and polymeric components were assembled to serve as vehicles for the transfection of messenger RNA (mRNA) using different portions of the cationic lipid DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and the cationic biopolymer protamine as model systems. Two different sequential assembly approaches in comparison with a direct single-step protocol were applied, and molecular organization in correlation with biological activity of the resulting nanoparticle systems was investigated. Differences in the structure of the nanoparticles were revealed by thorough physicochemical characterization including small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). All hybrid systems, combining lipid and polymer, displayed significantly increased transfection in comparison to lipid/mRNA and polymer/mRNA particles alone. For the hybrid nanoparticles, characteristic differences regarding the internal organization, release characteristics, and activity were determined depending on the assembly route. The systems with the highest transfection efficacy were characterized by a heterogenous internal organization, accompanied by facilitated release. Such a system could be best obtained by the single step protocol, starting with a lipid and polymer mixture for nanoparticle formation.

The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I interferon signaling pathway.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus causing the pandemic of severe pneumonia (Coronavirus Disease 2019, COVID-19). SARS-CoV-2 is highly pathogenic in human, having posed immeasurable public health challenges to the world. Innate immune response is critical for the host defense against viral infection and the dysregulation of the host innate immune responses probably aggravates SARS-CoV-2 infection, contributing to the high morbidity and lethality of COVID-19. It has been reported that some coronavirus proteins play an important role in modulating innate immunity of the host, but few studies have been conducted on SARS-CoV-2. In this study, we screened the viral proteins of SARS-CoV-2 and found that the viral ORF6, ORF8 and nucleocapsid proteins were potential inhibitors of type I interferon signaling pathway, a key component for antiviral response of host innate immune. All the three proteins showed strong inhibition on type I interferon (IFN-ß) and NF-κB-responsive promoter, further examination revealed that these proteins were able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus, while only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with interferon beta. These findings would be helpful for the further study of the detailed signaling pathway and unveil the key molecular player that may be targeted.