Effects of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers

<p>Abstract</p> <p>Background</p> <p>Efforts to improve the efficiency of non-viral gene delivery require a better understanding of delivery kinetics of DNA molecules into clinically relevant cells. Towards this goal, three DNA molecules were employed to investigate the effects of DNA properties on cellular delivery: a circular plasmid DNA (c-DNA), a linearized plasmid DNA (l-DNA) formulated by single-site digestion of c-DNA, and smaller linear gene cassette generated by PCR (pcr-DNA). Four non-viral gene carriers were investigated for DNA delivery: polyethyleneimine (PEI), poly-<it>L</it>-Lysine (PLL), palmitic acid-grafted PLL (PLL-PA), and Lipofectamine-2000™. Particle formation, binding and dissociation characteristics, and DNA uptake by rat bone marrow stromal cells were investigated.</p> <p>Results</p> <p>For individual carriers, there was no discernible difference in the morphology of particles formed as a result of carrier complexation with different DNA molecules. With PEI and PLL carriers, no difference was observed in the binding interaction, dissociation characteristics, and DNA uptake among the three DNA molecules. The presence of serum in cell culture media did not significantly affect the DNA delivery by the polymeric carriers, unlike other lipophilic carriers. Using PEI as the carrier, c-DNA was more effective for transgene expression as compared to its linear equivalent (l-DNA) by using the reporter gene for Enhanced Green Fluorescent Protein. pcr-DNA was the least effective despite being delivered into the cells to the same extent.</p> <p>Conclusion</p> <p>We conclude that the nature of gene carriers was the primary determinant of cellular delivery of DNA molecules, and circular form of the DNA was more effectively processed for transgene expression.</p

Editorial: Current Approaches to CRISPR/Cas9 Delivery

Editorial on the Research Topic Current approaches to CRISPR/Cas9 deliver

Prospects for RNAi Therapy of COVID-19

COVID-19 caused by the SARS-CoV-2 virus is a fast emerging disease with deadly consequences. The pulmonary system and lungs in particular are most prone to damage caused by the SARS-CoV-2 infection, which leaves a destructive footprint in the lung tissue, making it incapable of conducting its respiratory functions and resulting in severe acute respiratory disease and loss of life. There were no drug treatments or vaccines approved for SARS-CoV-2 at the onset of pandemic, necessitating an urgent need to develop effective therapeutics. To this end, the innate RNA interference (RNAi) mechanism can be employed to develop front line therapies against the virus. This approach allows specific binding and silencing of therapeutic targets by using short interfering RNA (siRNA) and short hairpin RNA (shRNA) molecules. In this review, we lay out the prospect of the RNAi technology for combatting the COVID-19. We first summarize current understanding of SARS-CoV-2 virology and the host response to viral entry and duplication, with the purpose of revealing effective RNAi targets. We then summarize the past experience with nucleic acid silencers for SARS-CoV, the predecessor for current SARS-CoV-2. Efforts targeting specific protein-coding regions within the viral genome and intragenomic targets are summarized. Emphasizing non-viral delivery approaches, molecular underpinnings of design of RNAi agents are summarized with comparative analysis of various systems used in the past. Promising viral targets as well as host factors are summarized, and the possibility of modulating the immune system are presented for more effective therapies. We place special emphasis on the limitations of past studies to propel the field faster by focusing on most relevant models to translate the promising agents to a clinical setting. Given the urgency to address lung failure in COVID-19, we summarize the feasibility of delivering promising therapies by the inhalational route, with the expectation that this route will provide the most effective intervention to halt viral spread. We conclude with the authors’ perspectives on the future of RNAi therapeutics for combatting SARS-CoV-2. Since time is of the essence, a strong perspective for the path to most effective therapeutic approaches are clearly articulated by the authors

Suppression of Human Coronavirus 229E Infection in Lung Fibroblast Cells via RNA Interference

Despite extensive efforts to repurpose approved drugs, discover new small molecules, and develop vaccines, COVID-19 pandemic is still claiming victims around the world. The current arsenal of antiviral compounds did not perform well in the past viral infections (e.g., SARS), which casts a shadow of doubt for use against the new SARS-CoV-2. Vaccines should offer the ultimate protection; however, there is limited information about the longevity of the generated immunity and the protection against possible mutations. This study uses Human Coronavirus 229E as a model coronavirus to test the hypothesis that effective delivery of virus-specific siRNAs to infected cells will result in lower viral load and reduced cell death. Two different categories of nucleic acid delivery systems, Peptide/Lipid-Associated Nucleic Acids (PLANAs) and lipophilic polymers, were investigated for their toxicity in human lung fibroblast cells and their ability to deliver specific siRNAs targeting Spike and Envelope proteins in order to prevent cell death in infected cells. Selected siRNAs were effectively delivered to human lung fibroblast cells with negligible toxicity. Cell death due to viral infection was significantly reduced with individual and combinatorial silencing of selected viral proteins. The combinatorial silencing of Spike and Envelope proteins restored the cell viability completely and eliminated plaques in the investigated system. Our cell culture data indicate promising results for the RNAi based approach as an alternative antiviral treatment

Fibronectin-modified surfaces for evaluating the influence of cell adhesion on sensitivity of leukemic cells to siRNA nanoparticles

Aim: This study aimed to create fibronectin (FN)-grafted polymeric surfaces to investigate the influence of leukemic cell adhesion on siRNA treatment. Materials & methods: FN was grafted on plasma-treated PTFE surfaces using chemical crosslinkers. Adhesion and growth of chronic myeloid leukemia K562 cells on modified surfaces were investigated. The silencing effect of siRNA/lipid-polymers nanoparticles on cells grown on FN-grafted surfaces was evaluated. Results: Crosslinker-mediated immobilization showed significant FN grafting on surfaces, which provided K562 cell adhesion and growth advantage. siRNA nanoparticle silencing was similarly effective on FN-adhered and suspension-growing K562 cells. Conclusion: This study provided initial data to develop a cell-adhesive system to investigate therapeutic effects on leukemic cells. The response of chronic myeloid leukemia cells to siRNA nanoparticles was independent on cell attachment

Editorial: Current approaches to CRISPR/Cas9 delivery

Editorial on the Research Topic Current approaches to CRISPR/Cas9 deliver

Effect of Nonviral Plasmid Delivered Basic Fibroblast Growth Factor and Low Intensity Pulsed Ultrasound on Mandibular Condylar Growth: A Preliminary Study

Objective. Basic fibroblast growth factor (bFGF) is an important regulator of tissue growth. Previous studies have shown that low intensity pulsed ultrasound (LIPUS) stimulates bone growth. The objective of this study was to evaluate the possible synergetic effect of LIPUS and local injection of nonviral bFGF plasmid DNA (pDNA) on mandibular growth in rats. Design. Groups were control, blank pDNA, bFGF pDNA, LIPUS, and bFGF pDNA + LIPUS. Treatments were performed for 28 days. Significant increase was observed in mandibular height and condylar length in LIPUS groups. MicroCT analysis showed significant increase in bone volume fraction in bFGF pDNA + LIPUS group. Histomorphometric analysis showed increased cell count and condylar proliferative and hypertrophic layers widths in bFGF pDNA group. Results. Current study showed increased mandibular condylar growth in either bFGF pDNA or LIPUS groups compared to the combined group that showed only increased bone volume fraction. Conclusion. It appears that there is an additive effect of bFGF + LIPUS on the mandibular growth

Contralateral Facial Innervation in Healthy Subjects and in Patients with Peripheral Facial Palsy

Background: We aimed to investigate the extent of the response of the orbicularis oris muscle to stimulation of the contralateral facial nerve both in patients with peripheral facial palsy (PFP) and in healthy subjects. Methods: EMG was performed at 2–6 weeks after the onset of PFP in the patient group and at any time in the healthy control group. We performed nerve conduction testing, electroneurography, and surface and needle EMG. Results: A total of 276 participants (patients/healthy controls: 218/58) were analyzed. The extent of the response of the contralateral orbicularis oris muscles to facial nerve stimulation was higher in healthy controls compared to that in the affected group. The response of the contralateral orbicularis oris muscles to stimulation of the paralyzed facial nerve was more extensive in those patients to whom glucocorticoid or physical therapy had been given. Cross-facial innervation in the orbicularis oris muscle extended up to 1.5 cm in one-third of healthy controls and was higher than that in those with PFP. Glucocorticoid or physical therapy seemed to improve cross-innervation in facial palsy. Conclusions: Our findings suggest that the stimulus leading to the contralateral muscular response is mediated through crossing axons rather than muscular fibers