Nucleic Acid Delivery by Solid Lipid Nanoparticles Containing Switchable Lipids: Plasmid DNA vs. Messenger RNA

The development of safe and effective nucleic acid delivery systems remains a challenge, with solid lipid nanoparticle (SLN)-based vectors as one of the most studied systems. In this work, different SLNs were developed, by combination of cationic and ionizable lipids, for delivery of mRNA and pDNA. The influence of formulation factors on transfection efficacy, protein expression and intracellular disposition of the nucleic acid was evaluated in human retinal pigment epithelial cells (ARPE-19) and human embryonic kidney cells (HEK-293). A long-term stability study of the vectors was also performed. The mRNA formulations induced a higher percentage of transfected cells than those containing pDNA, mainly in ARPE-19 cells; however, the pDNA formulations induced a greater protein production per cell in this cell line. Protein production was conditioned by energy-dependent or independent entry mechanisms, depending on the cell line, SLN composition and kind of nucleic acid delivered. Vectors containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as unique cationic lipid showed better stability after seven months, which improved with the addition of a polysaccharide to the vectors. Transfection efficacy and long-term stability of mRNA vectors were more influenced by formulation-related factors than those containing pDNA; in particular, the SLNs containing only DOTAP were the most promising formulations for nucleic acid delivery.This research was funded by the MCIU/AEI/FEDER, UE (RTI2018-098672-B-I00) and by the UPV/EHU (GIU17/032)

Novel Golden Lipid Nanoparticles with Small Interference Ribonucleic Acid for Substrate Reduction Therapy in Fabry Disease

Substrate reduction therapy (SRT) has been proposed as a new gene therapy for Fabry disease (FD) to prevent the formation of globotriaosylceramide (Gb3). Nanomedicines containing different siRNA targeted to Gb3 synthase (Gb3S) were designed. Formulation factors, such as the composition, solid lipid nanoparticles (SLNs) preparation method and the incorporation of different ligands, such as gold nanoparticles (GNs), protamine (P) and polysaccharides, were evaluated. The new siRNA–golden LNPs were efficiently internalized in an FD cell model (IMFE-1), with GNs detected in the cytoplasm and in the nucleus. Silencing efficacy (measured by RT-qPCR) depended on the final composition and method of preparation, with silencing rates up to 90% (expressed as the reduction in Gb3S-mRNA). GNs conferred a higher system efficacy and stability without compromising cell viability and hemocompatibility. Immunocytochemistry assays confirmed Gb3S silencing for at least 15 days with the most effective formulations. Overall, these results highlight the potential of the new siRNA–golden LNP system as a promising nanomedicine to address FD by specific SRT.This research was funded by MCIU/AEI/FEDER, UE, grant number RTI2018-098672-B-I00; by the UNIVERSITY OF THE BASQUE COUNTRY UPV/EHU, grant number GIU20/048; and by the BASQUE GOVERNMENT, grant number IT1587-22, GIC21/34

Galactomannan-Decorated Lipidic Nanocarrier for Gene Supplementation Therapy in Fabry Disease

Gene supplementation therapy with plasmid DNA (pDNA) represents one of the most promising strategies for the treatment of monogenic diseases such as Fabry disease (FD). In the present work, we developed a solid lipid nanoparticles (SLN)-based non-viral vector with a size below 100 nm, and decorated with galactomannan (GM) to target the liver as an α-Galactosidase A (α-Gal A) production factory. After the physicochemical characterization of the GM-SLN vector, cellular uptake, transfection efficacy and capacity to increase α-Gal A activity were evaluated in vitro in a liver cell line (Hep G2) and in vivo in an animal model of FD. The vector showed efficient internalization and it was highly efficient in promoting protein synthesis in Hep G2 cells. Additionally, the vector did not show relevant agglutination of erythrocytes and lacked hemolytic activity. After the systemic administration to Fabry mice, it achieved clinically relevant α-Gal A activity levels in plasma, liver, and other organs, importantly in heart and kidneys, two of the most damaged organs in FD. This work shows the potential application of GM-decorated lipidic nanocarries for the treatment of FD by pDNA-based gene augmentation.This research was funded by MCIU/AEI/FEDER, UE (RTI2018-098672-B-I00) and by the University of the Basque Country UPV/EHU (GIU20/048)

α-Galactosidase A Augmentation by Non-Viral Gene Therapy: Evaluation in Fabry Disease Mice

Fabry disease (FD) is a monogenic X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-Galactosidase A (α-Gal A). It is a good candidate to be treated with gene therapy, in which moderately low levels of enzyme activity should be sufficient for clinical efficacy. In the present work we have evaluated the efficacy of a non-viral vector based on solid lipid nanoparticles (SLN) to increase α-Gal A activity in an FD mouse model after intravenous administration. The SLN-based vector incremented α-Gal A activity to about 10%, 15%, 20% and 14% of the levels of the wild-type in liver, spleen, heart and kidney, respectively. In addition, the SLN-based vector significantly increased α-Gal A activity with respect to the naked pDNA used as a control in plasma, heart and kidney. The administration of a dose per week for three weeks was more effective than a single-dose administration. Administration of the SLN-based vector did not increase liver transaminases, indicative of a lack of toxicity. Additional studies are necessary to optimize the efficacy of the system; however, these results reinforce the potential of lipid-based nanocarriers to treat FD by gene therapy.This research was funded by Merck Salud Foundation, MCIU/AEI/FEDER, UE (RTI2018-098672-B-I00) and by the University of the Basque Country UPV/EHU (GIU17/032)

mRNA-Based Nanomedicinal Products to Address Corneal Inflammation by Interleukin-10 Supplementation

The anti-inflammatory cytokine Interleukin-10 (IL-10) is considered an efficient treatment for corneal inflammation, in spite of its short half-life and poor eye bioavailability. In the present work, mRNA-based nanomedicinal products based on solid lipid nanoparticles (SLNs) were developed in order to produce IL-10 to treat corneal inflammation. mRNA encoding green fluorescent protein (GFP) or human IL-10 was complexed with different SLNs and ligands. After, physicochemical characterization, transfection efficacy, intracellular disposition, cellular uptake and IL-10 expression of the nanosystems were evaluated in vitro in human corneal epithelial (HCE-2) cells. Energy-dependent mechanisms favoured HCE-2 transfection, whereas protein production was influenced by energy-independent uptake mechanisms. Nanovectors with a mean particle size between 94 and 348 nm and a positive superficial charge were formulated as eye drops containing 1% (w/v) of polyvinyl alcohol (PVA) with 7.1–7.5 pH. After three days of topical administration to mice, all formulations produced GFP in the corneal epithelium of mice. SLNs allowed the obtaining of a higher transfection efficiency than naked mRNA. All formulations produce IL-10, and the interleukin was even observed in the deeper layers of the epithelium of mice depending on the formulation. This work shows the potential application of mRNA-SLN-based nanosystems to address corneal inflammation by gene augmentation therapy.This research was funded by MCIU/AEI/FEDER, UE (SAF2014-53092-R), the UPV/EHU (GIU 20/048) and by the Università degli Studi di Torino (Ricerca Locale 2019)

Gene-terapia: ikuspegi terapeutiko berria begietako gaitzen tratamenduan

Gene-terapia etorkizun handiko tresna bezala sortu da tratamendurik ez duten asaldurentzat. Azido nukleiko terapeutikoen administrazioan oinarritzen da gaixotasunak tratatzeko. Gene-terapia arrakastatsua izan dadin material genetikoaren askapen eraginkorra bermatu behar da itu-zeluletan. Geneen administrazio-sistemen artean, bektore biralak asko erabili dira ahalbidetzen duten transferentzia genikorako gaitasun onarengatik. Hala ere, horien arrisku nagusien ondorioz (immunogenizitatea eta mutagenesia), bektore ez-biralen diseinua sustatu da. Bektore ez-biralak seguruagoak dira eta ekoizpena errazagoa da, baina hauen muga nagusia transfekzio-eraginkortasun baxua da. Gene-terapiarako organo interesgarri bat begia da, eskuragarria eta aztertzeko erraza baita. Gainera, immunitate-sistematik babestuta dago. Gene-terapia entsegu kliniko guztien % 1,3 bakarrik tratatzen dituzte begietako gaitzak, baina etorkizun handia aurkeztu dute azken urteetan. Izan ere, 2018an Estatu Batuetan eta Europan gene-terapian oinarritutako begirako lehen medikamentuaren komertzializazioa onartu zen, Luxturna®, Sortzetiko Leberren Amaurosiaren tratamendurako. Berrikuspen honetan, begiko administrazio-bideak, gene-terapia estrategiak eta transferentzia geniko eraginkorrerako gainditu behar diren begiko mugak aurkezten dira. Halaber, gene-terapiaren bidez tratatzeko hautagai diren begietako gaitz ezberdinak ere biltzen dira. Gene-terapiaren inguruan egindako ahaleginei eta aurrerapenei esker, begietako gaitzak tratatzeko medikamentu berriak garatu dira. Horietako asko entsegu klinikoetan ebaluatzen ari dira oraindik, baina beste batzuk jada merkatura eta pazienteetara iritsi dira.; Gene therapy has emerged as a promising tool for disorders that have no cure. It consists in the administration of therapeutic nucleic acids into patients for treating diseases. The success of gene therapy relies on the efficient delivery of the genetic material to target cells. Among gene delivery systems, viral vectors have been widely used due to their good gene transference efficacy. However, their potential risk associated with immunogenicity and mutagenesis has promoted the design of non-viral vectors. Non-viral vectors are safer and easier to produce, but their main limitation remains lower transfection efficacy. An attractive candidate for gene therapy is the eye, since it is easily accessible, easily examined and relatively immune privileged. Only 1,3% of all gene therapy clinical trials treat ocular disorders, but they have shown great potential in recent years. In fact, in 2018 the Food and Drug Administration and the European Medicine Agency approved the commercialization of the first gene therapy based ocular drug, Luxturna®, for the treatment of Leber Congenital Amaurosis. In this review, we present the main administration routes to eye, gene therapy strategies and ocular barriers to overcome for successful gene transfer. Different ocular disease candidates to be treated by gene therapy are also reviewed. The efforts and advances made in the field of gene therapy have led to the development of new drugs to treat eye diseases. Many of them are still being evaluated in clinical trials, but some have already reached the market and patients

Nanomedicines to Deliver mRNA: State of the Art and Future Perspectives

The use of messenger RNA (mRNA) in gene therapy is increasing in recent years, due to its unique features compared to plasmid DNA: Transient expression, no need to enter into the nucleus and no risk of insertional mutagenesis. Nevertheless, the clinical application of mRNA as a therapeutic tool is limited by its instability and ability to activate immune responses; hence, mRNA chemical modifications together with the design of suitable vehicles result essential. This manuscript includes a revision of the strategies employed to enhance in vitro transcribed (IVT) mRNA functionality and efficacy, including the optimization of its stability and translational efficiency, as well as the regulation of its immunostimulatory properties. An overview of the nanosystems designed to protect the mRNA and to overcome the intra and extracellular barriers for successful delivery is also included. Finally, the present and future applications of mRNA nanomedicines for immunization against infectious diseases and cancer, protein replacement, gene editing, and regenerative medicine are highlighted.This research was funded by the MCIU/AEI/FEDER, UE (RTI2018-098672-B-I00), and by the UPV/EHU (GIU17/032)

Topical Administration of SLN-Based Gene Therapy for the Treatment of Corneal Inflammation by De Novo IL-10 Production

One of the main challenges in gene therapy is the issue of delivery, and it is especially relevant for the success of gene therapy in the cornea. In the present work, eye drops containing biocompatible non-viral vectors based on solid lipid nanoparticles (SLNs) as gene delivery systems to induce the expression of interleukin 10 (IL-10) were designed to address the treatment of corneal inflammation. Two kinds of SLNs combined with different ligands (protamine, dextran, or hyaluronic acid (HA)) and formulated with polyvinyl alcohol (PVA) were prepared. SLN-based vectors were characterized in terms of size, adhesiveness, viscosity, and pH, before topical administration to wild type and IL-10 knock out (KO) mice. The formulations showed a homogenous particle size below 400 nm and a positive surface charge to favor bioadhesion; the incorporation of PVA improved the corneal penetration. After three days of treatment by topical instillation, SLN-based vectors mainly transfected corneal epithelial cells, HA-formulations being the most effective ones. IL-10 was capable of reaching even the endothelial layer. Corneal sections showed no histological change and formulations seemed to be well tolerated after repeated topical administration. These promising results highlight the possible contribution of non-viral gene augmentation therapy to the future clinical approach of corneal gene therapy.This research was funded by the Ministerio de Economía y Competitividad (SAF2014-53092-R), by FEDER funds from the EU, by the UPV/EHU (GIU17/032) and by the Università degli Studi di Torino (Ricerca Locale 2018)