Assembly strategy of liposome and polymer systems for siRNA delivery

In recent years, gene therapy has made tremendous progress in the development of disease treatment. Among them, siRNA offers specificity of gene silencing, ease of synthesis, and short development period, and has been intensively studied worldwide. However, siRNA as the hydrophilic polyanion is easily degraded in vivo and poorly taken up into cells and so, the benefits of its powerful gene silencing ability will not be realized until better carriers are developed that are capable of protecting siRNA and delivering it intact to the cytoplasm of the target cells. Cationic liposomes (CL) and cationic polymers (CP) are the main non-viral siRNA vectors, there have been a lot of reports on the use of these two carriers to deliver siRNA. Whereas, as far as we know, there have been few review articles that provide an in-depth summary of the siRNA loading principle and internal structures of the siRNA delivery system. We summarize the formation principle and assembly structure of the cationic liposome-siRNA and polymer-siRNA complexes, and point out their advantages and characteristics and also show how to perfect their assembly and improve their clinical application in the future. It supports some useful suggestions for siRNA therapy, specifically, safe and efficient delivery

Cystic fibrosis findings

CYSTIC FIBROSIS (CF) is one of the most common autosomal recessive genetic disorders. It occurs in approximately one in every 2,000 live births in the UK, and there are around 60,000 people with the condition worldwide. CF was first described in the 1930s; the name refers to the scarring (fibrosis) and cysts observed within the pancreas of sufferers. It is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP-activated chloride channel. The most severe effects of CF are in the lung, but the disorder also affects the pancreas, liver and intestines. It causes imbalanced water and ion movement across the epithelium of multiple organs which, in the periciliary liquid (PCL) layer of the lungs, produces a thick mucus. This is, in turn, associated with inflammation and bacterial infection, as well as life

MYCN Silencing by RNA Interference Induces Neurogenesis and Suppresses Proliferation in Models of Neuroblastoma with Resistance to Retinoic Acid

Neuroblastoma (NB) is the most common solid tumor in childhood. Twenty percent of patients display MYCN amplification, which indicates a very poor prognosis. MYCN is a highly specific target for an NB tumor therapy as MYCN expression is absent or very low in most normal cells, while, as a transcription factor, it regulates many essential cell activities in tumor cells. We aim to develop a therapy for NB based on MYCN silencing by short interfering RNA (siRNA) molecules, which can silence target genes by RNA interference (RNAi), a naturally occurring method of gene silencing. It has been shown previously that MYCN silencing can induce apoptosis and differentiation in MYCN amplified NB. In this article, we have demonstrated that siRNA-mediated silencing of MYCN in MYCN-amplified NB cells induced neurogenesis in NB cells, whereas retinoic acid (RA) treatment did not. RA can differentiate NB cells and is used for treatment of residual disease after surgery or chemotherapy, but resistance can develop. In addition, MYCN siRNA treatment suppressed growth in a MYCN-amplified NB cell line more than that by RA. Our result suggests that gene therapy using RNAi targeting MYCN can be a novel therapy toward MYCN-amplified NB that have complete or partial resistance toward RA

Genetic therapies for cystic fibrosis lung disease

Gene therapy for cystic fibrosis (CF) has been the subject ofintense research over the last twenty-five years or more, usingboth viral and liposomal delivery methods, but so far withoutthe emergence of a clinical therapy. New approaches to CFgene therapy involving recent improvements to vector systems,both viral and non-viral, as well as new nucleic acidtechnologies have led to renewed interest in the field. The fieldof therapeutic gene editing is rapidly developing with theemergence of CRISPR/Cas9 as well as chemically modifiedmRNA therapeutics. These new types of nucleic acid therapiesare also a good fit with delivery by non-viral deliveryapproaches which has led to a renewed interest in lipid-basedand other nanoformulation

A critical role for ATF2 transcription factor in the regulation of E-selectin expression in response to non-endotoxin components of Neisseria meningitidis

Vascular injury is a serious complication of sepsis due to the gram-negative bacterium Neisseria meningitidis. One of the critical early steps in initiating this injury is via the interaction of leucocytes, particularly neutrophils, with adhesion molecules expressed on inflamed endothelium. We have previously demonstrated that both lipopolysaccharide (LPS) and non-LPS components of meningococci can induce very high levels of expression of the vascular endothelial cell adhesion molecule E-selectin, which is critical for early tethering and capture of neutrophils onto endothelium under flow. Using an LPS-deficient strain of meningococcus, we showed that very high levels of expression can be induced in primary endothelial cells, even in the context of weak activation of the major host signal transduction factor [nuclear factor-κB (NF-κB)]. In this study, we show that the particular propensity for N. meningitidis to induce high levels of expression is regulated at a transcriptional level, and demonstrate a significant role for phosphorylation of the ATF2 transcription factor, likely via mitogen-activated protein (MAP) kinases, on the activity of the E-selectin promoter. Furthermore, inhibition of E-selectin expression in response to the lpxA- strain by a p38 inhibitor indicates a significant role of a p38-dependent MAPK signalling pathway in ATF2 activation. Collectively, these data highlight the role that LPS and other bacterial components have in modulating endothelial function and their involvement in the pathogenesis of meningococcal sepsis. Better understanding of these multiple mechanisms induced by complex stimuli such as bacteria, and the specific inflammatory pathways they activate, may lead to improved, focused interventions in both meningococcal and potentially bacterial sepsis more generally

New approaches to genetic therapies for cystic fibrosis

Gene therapy offers great promise for cystic fibrosis which has never been quite fulfilled due to the challenges of delivering sufficient amounts of the CFTR gene and expression persistence for a sufficient period of time in the lungs to have any effect. Initial trials explored both viral and non-viral vectors but failed to achieve a significant breakthrough. However, in recent years, new opportunities have emerged that exploit our increased knowledge and understanding of the biology of CF and the airway epithelium. New technologies include new viral and non-viral vector approaches to delivery, but also alternative nucleic acid technologies including oligonucleotides and siRNA approaches for gene silencing and gene splicing, described in this review, as presented at the 2019 annual European CF Society Basic Science meeting (Dubrovnik, Croatia). We also briefly discuss other emerging technologies including mRNA and CRISPR gene editing that are advancing rapidly. The future prospects for genetic therapies for CF are now diverse and more promising probably than any time since the discovery of the CF gene

The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations.

Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationic, receptor-targeting peptides have potential use in gene therapy applications. Lipopolyplex formulations described here are typically far more efficient transfection agents than binary lipoplex or polyplex formulations. It has been shown previously that the peptide component mediates both DNA packaging and targeting of the nanoparticle while in this report we investigate the contribution of the lipid component. We hypothesised that the lipid components synergise with the peptides in the transfection process by promoting endosomal escape after lipid bilayer fusion. Lipopolyplexes were prepared with cationic liposomes comprising DOTAP with either neutral lipid DOPE or DOPC. DOPE promotes fusogenic, inverted hexagonal lipid structures while DOPC promotes more stable laminar structures. Lipopolyplexes containing DOPE showed substantially higher transfection efficiency than those formulated with DOPC, both in vitro and in vivo. DOPE-containing lipopolyplexes showed rapid endosomal trafficking and nuclear accumulation of DNA while DOPC-containing formulations remained within the late endo-lysosomal compartments. These findings are consistent with previous finding for the role of DOPE in lipoplexes and support the hypothesis regarding the function of the lipid components in lipopolyplexes. These findings will help to inform future lipopolyplex design, strategies and clinical development processes

A Nanosensor Toolbox for Rapid, Label-Free Measurement of Airway Surface Liquid and Epithelial Cell Function

Ciliated lung epithelial cells and the airway surface liquid (ASL) comprise one of the body's most important protective systems. This system is finely tuned, and perturbations to ASL rheology, ASL depth, ASL pH, the transepithelial potential, and the cilia beat frequency are all associated with disease pathology. Further, these apparently distinct properties interact with each other in a complex manner. For example, changes in ASL rheology can result from altered mucin secretion, changes in ASL pH, or changes in ASL depth. Thus, one of the great challenges in trying to understand airway pathology is that the properties of the ASL/epithelial cell system need to be assessed near-simultaneously and without perturbing the sample. Here, we show that nanosensor probes mounted on a scanning ion conductance microscope make this possible for the first time, without any need for labeling. We also demonstrate that ASL from senescence-retarded human bronchial epithelial cells retains its native properties. Our results demonstrate that by using a nanosensor approach, it is possible to pursue faster, more accurate, more coherent, and more informative studies of ASL and airway epithelia in health and disease