Tumor Necrosis Factor (TNF) Related Apoptosis-Inducing Ligand (TRAIL), an immune cytokine of TNF-family, has received much attention in late 1990s as a potential cancer therapeutics due to its ...selective ability to induce apoptosis in cancer cells. TRAIL binds to cell surface death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) and facilitates formation of death-inducing signaling complex (DISC), eventually activating the p53-independent apoptotic cascade. This unique mechanism makes the TRAIL a potential anticancer therapeutic especially for p53-mutated tumors. However, recombinant human TRAIL protein (rhTRAIL) and TRAIL-R agonist monoclonal antibodies (mAb) failed to exert robust anticancer activities due to inherent and/or acquired resistance, poor pharmacokinetics and weak potencies for apoptosis induction. To get TRAIL back on track as a cancer therapeutic, multiple strategies including protein modification, combinatorial approach and TRAIL gene therapy are being extensively explored. These strategies aim to enhance the half-life and bioavailability of TRAIL and synergize with TRAIL action ultimately sensitizing the resistant and non-responsive cells. We summarize emerging strategies for enhanced TRAIL therapy in this review and cover a wide range of recent technologies that will provide impetus to rejuvenate the TRAIL therapeutics in the clinical realm.
Display omitted
Prospects for RNAi Therapy of COVID-19 Uludağ, Hasan; Parent, Kylie; Aliabadi, Hamidreza Montazeri ...
Frontiers in bioengineering and biotechnology,
07/2020, Letnik:
8
Journal Article
Recenzirano
Odprti dostop
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.
Abstract The discovery of RNA interference (RNAi) has excited the scientific field due to its potential for wide range of therapeutic applications. The pharmacological mediator of RNAi, short ...interfering RNA (siRNA), however, has faced significant obstacles in reaching its target site and effectively exerting its silencing activity. Effective pharmacological use of siRNA requires ‘carriers’ that can deliver the siRNA to its intended site of action. The carriers assemble the siRNA into supramolecular complexes that display functional properties during the delivery process. This review will summarize non-viral approaches to siRNA delivery, emphasizing the current obstacles to delivery and the mechanisms employed to overcome these obstacles. The carriers successfully pursued in pre-clinical (animal) models will be presented so as to provide a glimpse of possible candidates for clinical testing. Supramolecular assembly of nucleic acids with carriers will be probed from thermodynamics and computational perspectives to understand supramolecular structures and their dynamics. The delivery and trafficking requirements for siRNA are then dissected and engineering approaches to overcoming these barriers will be articulated. The latter has been attempted both at the cellular levels, focusing on intracellular barriers, as well as systemic level, emphasizing macroscopic challenges affecting siRNA delivery. Clinical experience with non-viral siRNA delivery is summarized, highlighting the nature delivery modes attempted in clinical settings. We conclude with a perspective on the future of siRNA therapeutics, specifically concentrating on the possible impact of non-viral carriers in the field.
The field of nanotechnology, which aims to control and utilize matter generally in 1-100 nm range, has been at the forefront of pharmaceutical development. Nanoparticulate delivery systems, with ...their potential to control drug release profiles, prolonging the presence of drugs in circulation, and to target drugs to a specific site, hold tremendous promise as delivery strategies for therapeutics. Growth factors are endogenous polypeptides that initiate intracellular signals to regulate cellular activities, such as proliferation, migration and differentiation. With improved understanding of their roles in physiopathology and expansion of their availability through recombinant technologies, growth factors are becoming leading therapeutic candidates for tissue engineering approaches. However, the outcome of growth factor therapeutics largely depends on the mode of their delivery due to their rapid degradation in vivo, and non-specific distribution after systemic administration. In order to overcome these impediments, nanoparticulate delivery systems are being harnessed for spatiotemporal controlled delivery of growth factors. This review presents recent advances and some disadvantages of various nanoparticulate systems designed for effective intact growth factor delivery. The therapeutic applications of growth factors delivered by such systems are reviewed, especially for bone, skin and nerve regeneration as well as angiogenesis. Finally, future challenges and directions in the field are presented in addition to the current limitations.
Recent advances in electrospinning are yielding intricate scaffolds for use in regenerative medicine. To explore the possibility of creating bioactive scaffolds with functional gene expression ...systems, electrospun gelatin mats bearing plasmid DNA (pDNA) polyplexes are explored. The pDNA is first condensed with a lipid-modified polyethylenimine (PEI) to create polyplexes including a poly(aspartic acid) (pAsp) additive, and subsequently electrospun after mixing the polyplexes in gelatin solution. The pDNA polyplexes, 82 nm in size with ζ-potential of +20 mV, are uniformly entrapped in mats with fiber diameter ranging between ~150 and ~350 nm. The additive complexes with pAsp display a significantly higher transfection activity in solution, which was also retained after entrapment in electrospun mats, based on GFP expression to human myoblast (C2C12) and mouse osteoblast cells (MC3T3-E1). Electrospinning of gelatin with polyethylene glycol improves the transfection efficiency, due to increased pDNA entrapment (~71%). To further validate gene-activated mats, a pDNA encoding BMP-2 shows robust alkaline phosphatase (ALP) induction in C2C12 and MC3T3-E1 cells as a marker of osteogenic differentiation. We conclude that creating gelatin fiber mats with bioactive pDNA polyplexes was feasible and such mats could aid in regenerative repair of a wide range of tissues.
Display omitted
Abstract In this study, an engineered non-viral polymer based delivery systems with structural features mimicking that of viral vectors was developed and the potential of this carrier for siRNA ...delivery was assessed. The developed siRNA carrier was based on poly(ethylene oxide)- block -poly(ε-caprolactone) (PEO- b -PCL) micelles decorated with integrin αvβ3 targeting peptide (RGD4C) and/or cell penetrating peptide (TAT) on the PEO shell, and modified with a polycation (spermine) in the PCL core for siRNA binding and protection. We observed increased cellular uptake and effective endosomal escape of siRNA delivered with the peptide-functionalized micelles especially those with dual functionality (RGD/TAT-micelles) compared to unmodified micelles (NON-micelles) in MDA435/LCC6 resistant cells. Transfection of mdr1 siRNA formulated in peptide-modified micelles led to P-gp down regulation both at the mRNA and protein level. Subsequent to P-gp down regulation, increased cellular accumulation of P-gp substrate, doxorubicin (DOX), in the cytoplasm and nucleus of resistant MDA435/LCC6 cells after treatment with peptide decorated polymeric micelle/mdr1 siRNA complexes was observed. As a result, resistance to DOX was successfully reversed. Interestingly, RGD/TAT-micellar siRNA complexes produced improved cellular uptake, P-gp silencing, DOX cellular accumulation, DOX nuclear localization and DOX induced cytotoxicity in MDA435/LCC6 cells when compared to micelles decorated with individual peptides. Results of this study indicated a potential for RGD/TAT-functionalized virus-like micelles as promising carriers for efficient delivery of mdr1 siRNA to MDA435/LCC6 resistant cells as means to reverse the P-gp mediated multidrug resistance to DOX.
Delivery of small interfering RNA (siRNA) for silencing of aberrantly expressed genes is a promising therapy for the treatment of various genetic disorders. Polymeric carriers have been used in the ...design of efficient delivery systems to generate nanoscale siRNA polyplexes. Despite the great amount of research pursued on siRNA therapeutics, the underlying mechanisms of polyplex dissociation in cytosol are still unclear. The fate of siRNA polyplexes during intracellular stages of delivery and how the endogenous molecules may affect the integrity of polyplexes remains to be explored. In this study, we have focused on miRNA-21 as a representative anionic endogenous molecule and performed gel electrophoresis mobility shift assays, particle size and zeta (ζ)-potential analyses, and a series of all-atom molecular dynamics simulations to elucidate the effect of miRNA on siRNA–PEI polyplexes. We report a slightly better binding to PEI by miRNA than that of siRNA, and speculated that miRNA may disrupt the integrity of preformed siRNA–PEI polyplexes. In contrast to our initial speculation, however, introduction of miRNA to a preformed siRNA–PEI polyplex revealed formation of a miRNA layer surrounding the polyplex through interactions with PEI. The resulting structure is a ternary siRNA–PEI–miRNA complex, where the experimentally determined ζ-potential was found to decrease as a function of miRNA added.
Abstract Hydrophobic modifications have emerged as a promising approach to improve the efficiency of non-viral gene delivery vectors (GDV). Functional GDVs from non-toxic polymers have been created ...with this approach but the mechanism(s) behind lipid-mediated enhancement in transfection remains to be clarified. Using a linoleic acid-substituted 2 kDa polyethylenimine (PEI2LA), we aimed to define the cellular uptake pathways and intracellular trafficking of plasmid DNA in normal human foreskin fibroblast cells. Several pharmacological compounds were applied to selectively inhibit uptake by clathrin-mediated endocytosis (CME), caveolin-mediated endocytosis (CvME) and macropinocytosis. We found that PEI2LA complexes were taken up predominantly through CME, and to a lesser extent by CvME. In contrast, its precursor molecule, PEI2 complexes was internalized primarily by CvME and macropinocytosis. The commonly used 25 kDa PEI 25 complexes utilized all endocytic pathways, suggesting its efficiency is derived from a different set of transfection pathways than PEI2LA. We further applied several endosome disruptive agents and found that hypertonic media enhanced the transfection of PEI2LA by 6.5-fold. We infer that lipid substitution changes the normal uptake pathways significantly and transfection with hydrophobically modified GDVs may be further enhanced by incorporating endosome disruptive elements into vector design.
High molecular weight (HMW) polyethylenimine (PEI) is one of the most versatile nonviral gene vectors that was extensively investigated over the past two decades. The cytotoxic profile of HMW PEI, ...however, encouraged a search for safer alternatives. Because of lack of cytotoxicity of low molecular weight (LMW) PEI, enhancing its performance via hydrophobic modifications has been pursued to this end. Since the performance of modified PEIs depends on the nature and extent of substituents, we systematically investigated the effect of hydrophobic modification of LMW (1.2 kDa) PEI with a short propionic acid (PrA). Moderate enhancements in PEI hydrophobicity resulted in enhanced cellular uptake of polyplexes and siRNA-induced silencing efficacy, whereas further increase in PrA substitution abolished the uptake as well as the silencing. We performed all-atom molecular dynamics simulations to elucidate the mechanistic details behind these observations. A new assembly mechanism was observed by the presence of hydrophobic PrA moieties, where PrA migrated to core of the polyplex. This phenomenon caused higher surface hydrophobicity and surface charge density at low substitutions, and it caused deleterious effects on surface hydrophobicity and cationic charge at higher substitutions. It is evident that an optimal balance of hydrophobicity/hydrophilicity is needed to achieve the desired polyplex properties for an efficient siRNA delivery, and our mechanistic findings should provide valuable insights for the design of improved substituents on nonviral carriers.