How Far Are Non-Viral Vectors to Come of Age and Reach Clinical Translation in Gene Therapy?

Efficient delivery of genetic material into cells is a critical process to translate gene therapy into clinical practice. In this sense, the increased knowledge acquired during past years in the molecular biology and nanotechnology fields has contributed to the development of different kinds of non-viral vector systems as a promising alternative to virus-based gene delivery counterparts. Consequently, the development of non-viral vectors has gained attention, and nowadays, gene delivery mediated by these systems is considered as the cornerstone of modern gene therapy due to relevant advantages such as low toxicity, poor immunogenicity and high packing capacity. However, despite these relevant advantages, non-viral vectors have been poorly translated into clinical success. This review addresses some critical issues that need to be considered for clinical practice application of non-viral vectors in mainstream medicine, such as efficiency, biocompatibility, long-lasting effect, route of administration, design of experimental condition or commercialization process. In addition, potential strategies for overcoming main hurdles are also addressed. Overall, this review aims to raise awareness among the scientific community and help researchers gain knowledge in the design of safe and efficient non-viral gene delivery systems for clinical applications to progress in the gene therapy field.

Non-viral vectors based on cationic niosomes and minicircle DNA technology enhance gene delivery efficiency for biomedical applications in retinal disorders.

Low transfection efficiency is a major challenge to overcome in non-viral approaches to reach clinical practice. Our aim was to explore new strategies to achieve more efficient non-viral gene therapies for clinical applications and in particular, for retinal diseases. Cationic niosomes and three GFP-encoding genetic materials consisting on minicircle (2.3 kb), its parental plasmid (3.5 kb) and a larger plasmid (5.5 kb) were combined to form nioplexes. Once fully physicochemically characterized, in vitro experiments in ARPE-19 retina epithelial cells showed that transfection efficiency of minicircle nioplexes doubled that of plasmids ones, maintaining good cell viability in all cases. Transfections in retinal primary cells and injections of nioplexes in rat retinas confirmed the higher capacity of cationic niosomes vectoring minicircle to deliver the genetic material into retina cells. Therefore, nioplexes based on cationic niosomes vectoring minicircle DNA represent a potential tool for the treatment of inherited retinal diseases.

Stem cell-based gene delivery mediated by cationic niosomes for bone regeneration.

Bone morphogenetic protein-7(BMP-7) plays a pivotal role in the transformation of mesenchymal stem cells (MSCs) into bone. However, its impact is hampered due to its short half-life. Therefore, gene therapy may be an interesting approach to deliver BMP-7 gene to D1-MSCs. In this manuscript we prepared and characterized niosomes based on cationic lipid 2,3-di(tetradecyloxy)propan-1-amine, combined with polysorbate 80 for gene delivery purposes. Niosomes were characterized and combined initially with pCMS-EGFP reporter plasmid, and later with pUNO1-hBMP-7 plasmid to evaluate osteogenesis differentiation. Additionally, specific blockers of most relevant endocytic pathways were used to evaluate the intracellular disposition of complexes. MSCs transfected with niosomes showed increased growth rate, enhanced alkaline phosphatase activity (ALP) and extracellular matrix deposition which suggested the formation of osteoblast-like cells. We concluded that hBMP-7-transfected MSCs could be considered not only as an effective delivery tool of hBMP-7, but also as proliferating and bone forming cells for bone regeneration.

Gene delivery to the lungs: pulmonary gene therapy for cystic fibrosis.

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder where the defective gene, the cystic fibrosis transmembrane conductance regulator (CFTR), is well identified. Moreover, the respiratory tract can be targeted through noninvasive aerosolized formulations for inhalation. Therefore, gene therapy is considered a plausible strategy to address this disease. Conventional gene therapy strategies rely on the addition of a correct copy of the CFTR gene into affected cells in order to restore the channel activity. In recent years, genome correction strategies have emerged, such as zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases. These gene editing tools aim to repair the mutated gene at its original genomic locus with high specificity. Besides, the success of gene therapy critically depends on the nucleic acids carriers. To date, several clinical studies have been carried out to add corrected copies of the CFTR gene into target cells using viral and non-viral vectors, some of them with encouraging results. Regarding genome editing systems, preliminary in vitro studies have been performed in order to repair the CFTR gene. In this review, after briefly introducing the basis of CF, we discuss the up-to-date gene therapy strategies to address the disease. The review focuses on the main factors to take into consideration when developing gene delivery strategies, such as the design of vectors and plasmid DNA, in vitro/in vivo tests, translation to human use, administration methods, manufacturing conditions and regulatory issues.

New Insights into Gene Delivery to Human Neuronal Precursor NT2 Cells: A Comparative Study between Lipoplexes, Nioplexes, and Polyplexes.

The transfection of human NTera2/D1 teratocarcinoma-derived cell line (or NT2 cells) represents a promising strategy for the delivery of exogenous proteins or biological agents into the central nervous system (CNS). The development of suitable nonviral vectors with high transfection efficiencies requires a profound knowledge of the whole transfection process. In this work, we elaborated and characterized in terms of size and zeta potential three different nonviral vectors: lipoplexes (144 nm; -29.13 mV), nioplexes (142.5 nm; +35.4 mV), and polyplexes (294.8 nm; +15.1 mV). We compared the transfection efficiency, cellular uptake, and intracellular trafficking of the three vectors in NT2 cell line. Lipoplexes exhibited the highest percentages of EGFP positive cells. The values obtained with polyplexes were lower compared to lipoplexes but higher than the percentages obtained with nioplexes. Cellular uptake results had a clear correlation with respect to the corresponding transfection efficiencies. Regarding the endocytosis mechanism, lipoplexes enter in the cell, mainly, via clathrin-mediated endocytosis (CME) while polyplexes via caveolae-mediated endocytosis (CvME). Nioplexes were discarded for this experiment due to their low cellular uptake. By simulating an artificial endosome, we demonstrated that the vectors were able to release the DNA cargo once inside the late endosome. The data collected from this assay showed that at 6 h the genetic material carried by polyplexes was still located in the late endosome, while DNA carried by lipoplexes was already in the nucleus. This result indicates a faster intracellular traffic of the lipid-based vectors. Overall, our work gives new insights into the transfection process of NT2 cells by different nonviral vectors as a first step in the development of ex vivo gene therapy platform.

Enduring high-efficiency in vivo transfection of neurons with non-viral magnetoparticles in the rat visual cortex for optogenetic applications.

This work demonstrates the successful long-term transfection in vivo of a DNA plasmid vector in rat visual cortex neurons using the magnetofection technique. The transfection rates reached values of up to 97% of the neurons after 30days, comparable to those achieved by viral vectors. Immunohistochemical treatment with anti-EGFP antibodies enhanced the detection of the EYFP-channelrhodopsin expression throughout the dendritic trees and cell bodies. These results show that magnetic nanoparticles offer highly efficient and enduring in vivo high-rate transfection in identified neurons of an adult mammalian brain and suggest that the magnetotechnique facilitates the introduction of large functional genetic material like channelrhodopsin with safe non-viral vectors using minimally invasive approaches. FROM THE CLINICAL EDITOR: Gene therapy may be one of the treatment modalities for neurological diseases in the future. The use of viral transfection remains a concern due to restrictions to the size limit of the genetic material able to be packed, as well as safety issues. In this work, the authors evaluated magnetoplexes as an alternative vehicle. The results showed very promising data in that these nanoparticles could offer high transfection efficiency.

Carbon-Based Nanostructures as Emerging Materials for Gene Delivery Applications.

Gene therapeutics are promising for treating diseases at the genetic level, with some already validated for clinical use. Recently, nanostructures have emerged for the targeted delivery of genetic material. Nanomaterials, exhibiting advantageous properties such as a high surface-to-volume ratio, biocompatibility, facile functionalization, substantial loading capacity, and tunable physicochemical characteristics, are recognized as non-viral vectors in gene therapy applications. Despite progress, current non-viral vectors exhibit notably low gene delivery efficiency. Progress in nanotechnology is essential to overcome extracellular and intracellular barriers in gene delivery. Specific nanostructures such as carbon nanotubes (CNTs), carbon quantum dots (CQDs), nanodiamonds (NDs), and similar carbon-based structures can accommodate diverse genetic materials such as plasmid DNA (pDNA), messenger RNA (mRNA), small interference RNA (siRNA), micro RNA (miRNA), and antisense oligonucleotides (AONs). To address challenges such as high toxicity and low transfection efficiency, advancements in the features of carbon-based nanostructures (CBNs) are imperative. This overview delves into three types of CBNs employed as vectors in drug/gene delivery systems, encompassing their synthesis methods, properties, and biomedical applications. Ultimately, we present insights into the opportunities and challenges within the captivating realm of gene delivery using CBNs.

Progress in Gelatin as Biomaterial for Tissue Engineering.

Tissue engineering has become a medical alternative in this society with an ever-increasing lifespan. Advances in the areas of technology and biomaterials have facilitated the use of engineered constructs for medical issues. This review discusses on-going concerns and the latest developments in a widely employed biomaterial in the field of tissue engineering: gelatin. Emerging techniques including 3D bioprinting and gelatin functionalization have demonstrated better mimicking of native tissue by reinforcing gelatin-based systems, among others. This breakthrough facilitates, on the one hand, the manufacturing process when it comes to practicality and cost-effectiveness, which plays a key role in the transition towards clinical application. On the other hand, it can be concluded that gelatin could be considered as one of the promising biomaterials in future trends, in which the focus might be on the detection and diagnosis of diseases rather than treatment.

Design and Validation of a Process Based on Cationic Niosomes for Gene Delivery into Novel Urine-Derived Mesenchymal Stem Cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are stem cells present in adult tissues. They can be cultured, have great growth capacity, and can differentiate into several cell types. The isolation of urine-derived mesenchymal stem cells (hUSCs) was recently described. hUSCs present additional benefits in the fact that they can be easily obtained noninvasively. Regarding gene delivery, nonviral vectors based on cationic niosomes have been used and are more stable and have lower immunogenicity than viral vectors. However, their transfection efficiency is low and in need of improvement. METHODS: We isolated hUSCs from urine, and the cell culture was tested and characterized. Different cationic niosomes were elaborated using reverse-phase evaporation, and they were physicochemically characterized. Then, they were screened into hUSCs for transfection efficiency, and their internalization was evaluated. RESULTS: GPxT-CQ at a lipid/DNA ratio of 5:1 (w/w) had the best transfection efficiency. Intracellular localization studies confirmed that nioplexes entered mainly via caveolae-mediated endocytosis. CONCLUSIONS: In conclusion, we established a protocol for hUSC isolation and their transfection with cationic niosomes, which could have relevant clinical applications such as in gene therapy. This methodology could also be used for creating cellular models for studying and validating pathogenic genetic variants, and even for performing functional studies. Our study increases knowledge about the internalization of tested cationic niosomes in these previously unexplored cells.

Biomaterials in cell microencapsulation.

The field of cell encapsulation is advancing rapidly. This cell-based technology permits the local and long-term delivery ofa desired therapeutic product reducing or even avoiding the need ofimmunosuppressant drugs. The choice of a suitable material preserving the viability and functionality of enclosed cells becomes fundamental if a therapeutic aim is intended. Alginate, which is by far the most frequently used biomaterial in the field of cell microencapsulation, has been demonstrated to be probably the best polymer for this purpose due to its biocompatibility, easy manipulation, gel forming capacity and in vivo performance.

A study of the toxic effect of oxidized sunflower oil containing 4-hydroperoxy-2-nonenal and 4-hydroxy-2-nonenal on cortical TrkA receptor expression in rats.

Sunflower oil at a specific oxidation stage (when several oxygenated alpha,beta-unsaturated aldehydes are generated, mainly 4-hydroperoxy-trans-2-alkenals and 4-hydroxy-trans-2-alkenals), caused at 70 degrees C with aeration for 7 days, was administered intraperitoneally to rats. This oil was studied by means of solid phase micro-extraction followed by gas chromatography/mass spectrometry (SPME-GC/MS) and by proton nuclear magnetic resonance ((1)H-NMR). Oxidized sunflower oil (3 ml/kg/day) was administered to male Sprague-Dawley rats for 21 days. The control group was administered non-oxidized sunflower oil in the same volume and for the same duration as the experimental group. A significant decrease in the number of neural cells positively immunostained for TrkA receptor was detected in the frontal cortex of the experimental group, with respect to controls, suggesting both neuronal damage as well as a deficit in neuronal survival signalling at this level. This could lead to apoptosis of cholinergic neurons, which play a key role in memory and attention function. These results indicate that toxic substances present in the oxidized sunflower oil, among them 4-hydroxy-trans-2-nonenal (HNE) and 4-hydroperoxy-trans-2-nonenal (HPNE), could disrupt survival signalling of frontal cortex cholinergic neurons, which could lead to apoptosis and neurodegenerative diseases. In the case of humans, this fact reinforces the necessity of avoiding the re-utilization of oxidized sunflower oil, in order to contribute to long-term neurodegenerative diseases prevention.

Cationic Niosomes as Non-Viral Vehicles for Nucleic Acids: Challenges and Opportunities in Gene Delivery.

Cationic niosomes have become important non-viral vehicles for transporting a good number of small drug molecules and macromolecules. Growing interest shown by these colloidal nanoparticles in therapy is determined by their structural similarities to liposomes. Cationic niosomes are usually obtained from the self-assembly of non-ionic surfactant molecules. This process can be governed not only by the nature of such surfactants but also by others factors like the presence of additives, formulation preparation and properties of the encapsulated hydrophobic or hydrophilic molecules. This review is aimed at providing recent information for using cationic niosomes for gene delivery purposes with particular emphasis on improving the transportation of antisense oligonucleotides (ASOs), small interference RNAs (siRNAs), aptamers and plasmids (pDNA).

Brain endocannabinoid system is involved in fluoxetine-induced anorexia.

In order to describe the effects of chronic fluoxetine administration on the brain endocannabinoid system in lean and obese Zucker rats, brain immunostaining for the CB1 and CB1-phosphorylated cannabinoid receptors was carried out. Obese Zucker rats showed significantly increased the numbers of neural cells positively immunostained for the CB1-phosphorylated receptor in the striatum, compared to their lean litter-mates. Chronic fluoxetine administration decreased the number of neural cells immunostained for CB1-phosphorylated receptor in several striatal and hippocampal regions of obese Zucker rats, compared to controls treated with saline. In contrast, no change in CB1-phosphorylated receptor immunostaining was observed in fluoxetine-treated lean rats, with respect to controls. Taken together, these results suggest the involvement of the hippocampal and striatal endocannabinoid receptor system in fluoxetine-induced anorexia in lean and obese Zucker rats.

Hyaluronic acid hydrogel scaffolds loaded with cationic niosomes for efficient non-viral gene delivery.

The lack of ideal non-viral gene carriers has motivated the combination of delivery systems and tissue-engineered scaffolds, which may offer relevant advantages such as enhanced stability and reduced toxicity. In this work, we evaluated a new combination between niosome non-viral vectors and hyaluronic acid (HA) hydrogel scaffolds, both widely studied due to their biocompatibility as well as their ability to incorporate a wide variety of molecules. We evaluated three different niosome formulations (niosomes 1, 2 and 3) varying in composition of cationic lipid, helper lipid and non-ionic tensioactives. Niosomes and nioplexes obtained upon the addition of plasmid DNA were characterized in terms of size, polydispersity, zeta potential and ability to transfect mouse bone marrow cloned mesenchymal stem cells (mMSCs) in 2D culture. Niosome 1 was selected for encapsulation in HA hydrogels due to its higher transfection efficiency and the formulation was concentrated in order to be able to incorporate higher amounts of DNA within HA hydrogels. Nioplex-loaded HA hydrogels were characterized in terms of biomechanical properties, particle distribution, nioplex release kinetics and ability to transfect encapsulated mMSCs in 3D culture. Our results showed that nioplex-loaded HA hydrogel scaffolds presented little or no particle aggregation, allowed for extensive cell spreading and were able to efficiently transfect encapsulated mMSCs with high cellular viability. We believe that the knowledge gained through this in vitro model can be utilized to design novel and effective platforms for in vivo local and non-viral gene delivery applications.

Polysorbate 20 non-ionic surfactant enhances retinal gene delivery efficiency of cationic niosomes after intravitreal and subretinal administration.

The success of non-viral vectors based on cationic niosomes for retinal gene delivery applications depends on the ability to achieve persistent and high levels of transgene expression, ideally from a single administration. In this work, we studied the effect of the non-ionic surfactant component of niosomes in their transfection efficiency in rat retina. For that purpose, three niosome formulations that only differed in the non-ionic tensioactives were elaborated. Niosomes contained: cationic lipid 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), helper lipid squalene and polysorbate 20, polysorbate 80 or polysorbate 85. Niosomes and corresponding nioplexes were fully characterized in terms of size, polydispersity index, zeta potential, morphology and ability to protect and release DNA. In vitro experiments were carried out to evaluate transfection efficiency, cell viability and intracellular trafficking pathways of the formulations. Nioplexes based on polysorbate 20 niosomes were the most efficient transfecting retinal cells in vitro. Moreover, subretinal and intravitreal administration of those nioplexes in vivo showed also high levels of transgene expression in rat retinas. Our results demonstrate that the incorporation of polysorbate 20 in cationic niosomes enhances retinal gene delivery. Thus, this formulation emerges as a potential non-viral candidate to efficiently transfer specific therapeutic genes into the eye for biomedical purposes.

Cationic nioplexes-in-polysaccharide-based hydrogels as versatile biodegradable hybrid materials to deliver nucleic acids.

Two polysaccharide-based hydrogels made of only κ-carrageenan (4%; w/v) or of a mixture of methylcellulose:κ-carrageenan (2%; w/v) were used to encapsulate cationic nioplexes. These vesicular particles were made of a synthetic aminolipid and polysorbate-80 (Tween-80), as a non-ionic surfactant agent. According to oscillatory rheological measurements, the presence of nioplexes did not compromise the mechanical integrity of the gels. In vitro niosomal release experiments demonstrated the liberation of nioplexes up to 24 h, and the curves were fitted according to Higuchi, Korsmeyer-Peppas and Weibull equation models, which indicated Fickian-diffusion controlled mechanisms. Besides nioplexes, cervical cancer cells were also entrapped within the biohydrogels. Cell release confirmed that these materials did not affect the cell viability, allowing cells to spread and proliferate after 24 h. The applicability of these biocompatible hydrogels was also extended to gene delivery. In this regard, the best silencing activities were found when cationic niosomes were complexed with antisense oligonucleotides in KC hydrogels. Nioplexes were able to release through the hydrogel and promoted silencing of luciferase expression in the presence of serum without using commercially available cationic lipids. Overall, the formation of such hybrid materials by integrating cationic nioplexes within biodegradable hydrogels provides a new perspective for the delivery of macromolecular therapeutics.

Non-viral vectors based on magnetoplexes, lipoplexes and polyplexes for VEGF gene delivery into central nervous system cells.

Nanotechnology based non-viral vectors hold great promise to deliver therapeutic genes into the central nervous system (CNS) in a safe and controlled way. Vascular endothelial growth factor (VEGF) is a potential therapeutic gene candidate for CNS disorders due to its specific roles in brain angiogenesis and neuroprotection. In this work, we elaborated three different non-viral vectors based on magnetic, cationic lipid and polymeric nanoparticles complexed to the phVEGF165aIRESGFP plasmid, which codifies the VEGF protein -extracellular- and the green fluorescent protein (GFP) -intracellular-. Nanoparticles and corresponding nanoplexes -magnetoplexes, lipoplexes and polyplexes- were characterized in terms of size, zeta potential, polydispersity index, morphology and ability to bind, release and protect DNA. Transfection efficiencies of nanoplexes were measured in terms of percentage of GFP expressing cells, mean fluorescent intensity (MFI) and VEGF (ng/ml) production in HEK293, C6 and primary neuronal culture cells. Magnetoplexes showed the highest transfection efficiencies in C6, followed by lipoplexes, and in primary neuronal culture cells, followed by polyplexes. Lipoplexes were the most efficient in HEK293 cells, followed by magnetoplexes. The biological activity of VEGF was confirmed by its proliferative effect in HUVEC cells. Overall, these results provide new insights for VEGF gene delivery into CNS cells using non-viral vectors.

Elaboration and Physicochemical Characterization of Niosome-Based Nioplexes for Gene Delivery Purposes.

Niosome formulations for gene delivery purposes are based on nonionic surfactants, helper lipids, and cationic lipids that interact electrostatically with negatively charged DNA molecules to form the so-called nioplexes. Niosomes are elaborated by different techniques, such as solvent emulsion-evaporation, thin film hydration, hand-shaking, dissolvent injection, and microfluidization method, among many others. In this chapter, we have described some protocols for the elaboration of niosomes and nioplexes and their physicochemical characterization that guarantees the quality criteria of the formulation in terms of size, morphology, ζ-potential, and stability.

The role of helper lipids in the intracellular disposition and transfection efficiency of niosome formulations for gene delivery to retinal pigment epithelial cells.

In this work, we carried out a comparative study of four different niosome formulations based on the same cationic lipid and non-ionic tensoactive. The niosomes prepared by oil-in-water emulsion technique (o/w) only differed in the helper lipid composition: squalene, cholesterol, squalane or no helper lipid. Niosomes and nioplexes elaborated upon the addition of pCMS-EGFP reporter plasmid were characterized in terms of size, zeta potential and polydispersity index. The capacity of the niosomes to condense, release and protect the DNA against enzymatic degradation was evaluated by agarose gel electrophoresis. In vitro experiments were carried out to evaluate transfection efficiency and cell viability in retinal pigment epithelial cells. Moreover, uptake and intracellular trafficking studies were performed to further understand the role of the helper lipids in the transfection process. Interestingly, among all tested formulations, niosomes elaborated with squalene as helper lipid were the most efficient transfecting cells. Such transfection efficiency could be attributed to their higher cellular uptake and the particular entry pathways used, where macropinocytosis pathway and lysosomal release played an important role. Therefore, these results suggest that helper lipid composition is a crucial step to be considered in the design of niosome formulation for retinal gene delivery applications since clearly modulates the cellular uptake, internalization mechanism and consequently, the final transfection efficiency.

Improving transfection efficiency of ultrapure oligochitosan/DNA polyplexes by medium acidification.

CONTEXT: Ultrapure oligochitosans (UOCs) have recently been reported as efficient nonviral vectors for corneal and retinal gene delivery. However, the influence of some physicochemical factors on the transfection efficiency, such as the pH, remains unclear. Deeper in vitro research of these factors could provide valuable information for future clinical applications. OBJECTIVE: The aim of this study is to determine the influence of the pH decrease on the transfection efficiency of UOC/pDNA polyplexes in HEK293 and ARPE19 cells. MATERIALS AND METHODS: We elaborated self-assembled UOC/pCMS-EGFP polyplexes. The influence of the most important factors on the particle size and the zeta potential was studied by an orthogonal experimental design. We evaluated, in vitro, the cellular uptake and the transfection efficiency by flow cytometry, and the cytotoxicity of the vectors by CCK-8 assay. RESULTS AND DISCUSSION: The pH of the medium strongly influences the physicochemical properties of the polyplexes, and by its modulation we are able to control their superficial charge. A significant increase on the cellular uptake and transfection efficiency of UOCs was obtained when the pH was acidified. Neither of our UOC/pCMS-EGFP polyplexes caused cytotoxicity; however, cells treated with Lipofectamine 2000™ showed decreased cell viability. CONCLUSION: This kind of UOC vectors could be useful to transfect cells that are in an acidic environment, such as tumor cells. However, additional in vivo studies may be required in order to obtain an effective and safe medicine for nonviral gene therapy purpose.