Targeting strategies with lipid vectors for nucleic acid supplementation therapy in Fabry disease: a systematic review.

Fabry disease (FD) results from a lack of activity of the lysosomal enzyme α-Galactosidase A (α-Gal A), leading to the accumulation of glycosphingolipids in several different cell types. Protein supplementation by pDNA or mRNA delivery presents a promising strategy to tackle the underlying genetic defect in FD. Protein-coding nucleic acids in FD can be either delivered to the most affected sites by the disease, including heart, kidney and brain, or to specialized organs that can act as a production factory of the enzyme, such as the liver. Lipid-based systems are currently at the top of the ranking of non-viral nucleic acid delivery systems, and their versatility allows the linking to the surface of a wide range of molecules to control their biodistribution after intravenous administration. This systematic review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement guidelines and provides an overview and discussion of the targeting ligands that have been employed so far to actively vectorize intravenously administered non-viral vectors based on lipid carriers to clinically relevant organs in the treatment of FD, for protein-coding nucleic acid (pDNA and mRNA) supplementation. Among the thirty-two studies included, the majority focus on targeting the liver and brain. The targeting of the heart has been reported to a lesser degree, whereas no articles addressing kidney-targeting have been recorded. Although a great effort has been made to develop organ-specific nucleic acid delivery systems, the design of active-targeted carriers with high quality, good clinical translation, and large-scale manufacturing capacity is still challenging.

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.

Pharmacokinetic/Pharmacodynamic Analysis of Oral Calcium Fosfomycin: Are Urine Levels Sufficient to Ensure Efficacy for Urinary Tract Infections?

Urinary tract infections (UTIs) are extremely common and a major driver for the use of antimicrobials. Calcium fosfomycin is an old antibiotic indicated for the treatment of UTIs; however, data about its urine pharmacokinetic profile are scarce. In this work, we have evaluated the pharmacokinetics of fosfomycin from urine concentrations after oral administration of calcium fosfomycin to healthy women. Moreover, we have assessed, by pharmacokinetic/pharmacodynamic (PK/PD) analysis and Monte Carlo simulations, its effectiveness considering the susceptibility profile of Escherichia coli, the main pathogen involved in UTIs. The accumulated fraction of fosfomycin excreted in urine was around 18%, consistent with its low oral bioavailability and its almost exclusively renal clearance by glomerular filtration as unchanged drug. PK/PD breakpoints resulted to be 8, 16, and 32 mg/L for a single dose of 500 mg, a single dose of 1000 mg, and 1000 mg q8h for 3 days, respectively. For empiric treatment, the estimated probability of treatment success was very high (>95%) with the three dose regimens, considering the susceptibility profile of E. coli reported by EUCAST. Our results show that oral calcium fosfomycin at a dose level of 1000 mg every 8 h provides urine concentrations sufficient to ensure efficacy for the treatment of UTIs in women.

mRNA delivery technologies: Toward clinical translation.

Messenger RNA (mRNA)-therapies have recently taken a huge step toward clinic thanks to the first mRNA-based medicinal products marketed. mRNA features for clinical purposes are improved by chemical modifications, but the inclusion in a delivery system is a regular requirement. mRNA nanomedicines must be designed for the specific therapeutic purpose, protecting the nucleic acid and facilitating the overcoming of biological barriers. Polymers, polypeptides, and cationic lipids are the main used materials to design mRNA delivery systems. Among them, lipid nanoparticles (LNPs) are the most advanced ones, and currently they are at the forefront of preclinical and clinical evaluation in several fields, including immunotherapy (against infectious diseases and cancer), protein replacement, gene editing and regenerative medicine. This chapter includes an overview on mRNA delivery technologies, with special interest in LNPs, and the most recent advances in their clinical application. Liposomes are the mRNA delivery technology with the highest clinical translation among LNPs, whereas the first clinical trial of a therapeutic mRNA formulated in exosomes has been recently approved for protein replacement therapy. The first mRNA products approved by the regulatory agencies worldwide are LNP-based mRNA vaccines against viral infections, specifically against the 2019 coronavirus disease (COVID-19). The clinical translation of mRNA-therapies for cancer is mainly focused on three strategies: anti-cancer vaccination by means of delivering cancer antigens or acting as an adjuvant, mRNA-engineered chimeric antigen receptors (CARs) and T-cell receptors (TCRs), and expression of antibodies and immunomodulators. Cancer immunotherapy and, more recently, COVID-19 vaccines spearhead the advance of mRNA clinical use.

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.

Oferta de servicios de atención farmacéutica: clave para un nuevo modelo de servicios de salud / Supply of pharmaceutical care services: The key to a new model of health services

La colaboración interdisciplinar entre profesionales sanitarios es esencial para mejorar los resultados en salud de la población. Las capacidades que poseen los farmacéuticos les convierten en profesionales que pueden contribuir al control integral de la farmacoterapia en coordinación con otros profesionales de la salud. El Consejo de Europa aprobó la resolución CM/Res(2020)3 sobre implementación de la atención farmacéutica en el sistema sanitario para promover el uso apropiado y seguro de los medicamentos. Entre las actividades a realizar mediante el proceso de atención farmacéutica destaca la detección de problemas relacionados con la farmacoterapia como son las contraindicaciones, duplicidades, errores de prescripción, interacciones, etc. La intervención farmacéutica en ese ámbito requiere establecer un marco de colaboración interprofesional adecuado. En el presente artículo se comentan los aspectos a abordar para afrontar el cambio de modelo hacia una farmacia asistencial, con una mayor integración e implicación en el sistema, todo ello bajo el amparo del Consejo de Europa.

Interdisciplinary collaboration between health professionals is essential to improve health outcomes. The competences of pharmacists make them professionals capable of contributing to the comprehensive management of pharmacotherapy in collaboration with other healthcare professionals. The Council of Europe adopted resolution CM/Res(2020)3 on implementation of pharmaceutical care for the benefit of patients and health services in order to promote the appropriate and safe use of medicines. One of the activities to be carried out through the pharmaceutical care process is the detection of drug related problems, such as contraindications, duplications, prescription errors, interactions, etc. In many cases, it involves regular patient follow-up and needs an evaluation of the interventions performed, requiring the establishment of an appropriate interprofessional collaboration framework. This article discusses the issues to be addressed to face the change of model towards a care-based pharmacy.

[Supply of pharmaceutical care services: The key to a new model of health services]. / Oferta de servicios de atención farmacéutica: clave para un nuevo modelo de servicios de salud.

Interdisciplinary collaboration between health professionals is essential to improve health outcomes. The competences of pharmacists make them professionals capable of contributing to the comprehensive management of pharmacotherapy in collaboration with other healthcare professionals. The Council of Europe adopted resolution CM/Res(2020)3 on implementation of pharmaceutical care for the benefit of patients and health services in order to promote the appropriate and safe use of medicines. One of the activities to be carried out through the pharmaceutical care process is the detection of drug related problems, such as contraindications, duplications, prescription errors, interactions, etc. In many cases, it involves regular patient follow-up and needs an evaluation of the interventions performed, requiring the establishment of an appropriate interprofessional collaboration framework. This article discusses the issues to be addressed to face the change of model towards a care-based pharmacy.

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.

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.

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.

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.

Gene Therapy.

Gene therapy medicinal products (GTMPs) are one of the most promising biopharmaceuticals, which are beginning to show encouraging results. The broad clinical research activity has been addressed mainly to cancer, primarily to those cancers that do not respond well to conventional treatment. GTMPs to treat rare disorders caused by single-gene mutations have also made important advancements toward market availability, with eye and hematopoietic system diseases as the main applications.Nucleic acid-marketed products are based on both in vivo and ex vivo strategies. Apart from DNA-based therapies, antisense oligonucleotides, small interfering RNA, and, recently, T-cell-based therapies have been also marketed. Moreover, the gene-editing tool CRISPR is boosting the development of new gene therapy-based medicines, and it is expected to have a substantial impact on the gene therapy biopharmaceutical market in the near future.However, despite the important advancements of gene therapy, many challenges have still to be overcome, which are discussed in this book chapter. Issues such as efficacy and safety of the gene delivery systems and manufacturing capacity of biotechnological companies to produce viral vectors are usually considered, but problems related to cost and patient affordability must be also faced to ensure the success of this emerging therapy. Graphical Abstract.

MMP-9 Downregulation with Lipid Nanoparticles for Inhibiting Corneal Neovascularization by Gene Silencing.

Gene silencing targeting proangiogenic factors have been shown to be a useful strategy in the treatment of corneal neovascularization (CNV). Among interference RNA (RNAi) molecules, short-hairpin RNA (shRNA) is a plasmid-coded RNA able to down-regulate the expression of the desired gene. It is continuously produced in the host cell, inducing a durable gene silencing effect. The aim of this work was to develop a solid lipid nanoparticle (SLN)-based shRNA delivery system to downregulate metalloproteinase 9 (MMP-9), a proangiogenic factor, in corneal cells for the treatment of CNV associated with inflammation. The nanovectors were prepared using a solvent emulsification-evaporation technique, and after physicochemical evaluation, they were evaluated in different culture cell models. Transfection efficacy, cell internalization, cell viability, the effect on MMP-9 expression, and cell migration were evaluated in human corneal epithelial cells (HCE-2). The inhibition of tube formation using human umbilical vein endothelial cells (HUVEC) was also assayed. The non-viral vectors based on SLN were able to downregulate the MMP-9 expression in HCE-2 cells via gene silencing, and, consequently, to inhibit cell migration and tube formation. These results demonstrate the potential of lipid nanoparticles as gene delivery systems for the treatment of CNV-associated inflammation by RNAi technology.

Targeting corneal inflammation by gene therapy: Emerging strategies for keratitis.

Inflammation is the underlying process of several diseases within the eye, specifically in the cornea. Current treatment options for corneal inflammation or keratitis, and related neovascularization, are restricted by limited efficacy, adverse effects, and short duration of action. Gene therapy has shown great potential for the treatment of diseases affecting the ocular surface, and major efforts are being targeted to inflammatory mediators and neovascularization, in order to develop potential treatments for corneal inflammation. Gene therapy to treat ocular disorders is still starting, and current therapies are primarily experimental, with most human clinical trials still in research state, although some of them have already shown encouraging results. In this review, we focus on the progress and challenges of gene therapy to treat corneal inflammation. After introducing the inflammation process, we present the main nucleic acid delivery systems, including viral and non-viral vectors, and the most studied strategies to address the therapy: control of neovascularization and regulation of pro- and anti-inflammatory cytokines.

Applications of lipid nanoparticles in gene therapy.

Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) have been recognized, among the large number of non-viral vectors for gene transfection, as an effective and safety alternative to potentially treat both genetic and not genetic diseases. A key feature is the possibility to be designed to overcome the numerous challenges for successful gene delivery. Lipid nanoparticles (LNs) are able to overcome the main biological barriers for cell transfection, including degradation by nucleases, cell internalization intracellular trafficking, and selectively targeting to a specific cell type. Additionally, they present important advantages: from a safety point of view LNs are prepared with well tolerated components, and from a technological point of view, they can be easily produced at large-scale, can be subjected to sterilization and lyophilization, and have shown good storage stability. This review focuses on the potential of SLNs and NLCs for gene therapy, including the main advances in their application for the treatment of ocular diseases, infectious diseases, lysosomal storage disorders and cancer, and current research for their future clinical application.

Silencing of hepatitis C virus replication by a non-viral vector based on solid lipid nanoparticles containing a shRNA targeted to the internal ribosome entry site (IRES).

Gene silencing mediated by RNAi has gained increasing interest as an alternative for the treatment of infectious diseases such as refractory hepatitis C virus (HCV) infection. In this work we have designed and evaluated a non-viral vector based on solid lipid nanoparticles (SLN) bearing hyaluronic acid, protamine and a short hairpin RNA (shRNA74) targeted to the Internal Ribosome Entry Site (IRES) of the HCV. The vector was able to inhibit the expression of the HCV IRES in Huh-7 cells, with the inhibition level dependent on the shRNA74 to SLN ratio and on the shRNA74 dose added to the culture cells. The nanocarrier was also able to inhibit the replication in human hepatoma cells supporting a subgenomic HCV replicon (Huh-7 NS3-3'). The vector was quickly and efficiently internalized by the cells, and endocytosis was the most productive uptake mechanism for silencing. Clathrin-mediated endocytosis and to a lesser extent caveolae/lipid raft-mediated endocytosis were identified as endocytic mechanisms involved in the cell uptake. Internalization via the CD44 receptor was also involved, although this entry route seems to be less productive for silencing than endocytosis. The vector did not induce either hemolysis or agglutination of red cells in vitro, which was indicative of good biocompatibility. In summary, we have shown for the first time the ability of a non-viral SLN-based vector to silence a HCV replicon.

Application of lipid nanoparticles to ocular drug delivery.

INTRODUCTION: Although eye drops are widely used as drug delivery systems for the anterior segment of the eye, they are also associated with poor drug bioavailability due to transient contact time and rapid washout by tearing. Moreover, effective drug delivery to the posterior segment of the eye is challenging, and alternative routes of administration (periocular and intravitreal) are generally needed, the blood-retinal barrier being the major obstacle to systemic drug delivery. Areas covered: Nanotechnology, and especially lipid nanoparticles, can improve the therapeutic efficiency, compliance and safety of ocular drugs, administered via different routes, to both the anterior and posterior segment of the eye. This review highlights the main ocular barriers to drug delivery, as well as the most common eye diseases suitable for pharmacological treatment in which lipid nanoparticles have proved efficacious as alternative delivery systems. Expert opinion: Lipid-based nanocarriers are among the most biocompatible and versatile means for ocular delivery. Mucoadhesion with consequent increase in pre-corneal retention time, and enhanced permeation due to cellular uptake by corneal epithelial cells, are the essential goals for topical lipid nanoparticle delivery. Gene delivery to the retina has shown very promising results after intravitreal administration of lipid nanoparticles as non-viral vectors.

Vaginal gene therapy.

In the last years, vaginal gene therapy has gained increasing attention mainly for the treatment and control of sexually transmitted infections. DNA delivery has been also suggested to improve reproductive outcomes for women with deficiencies in the female reproductive tract. Although no product has reached clinical phase, preclinical investigations reveal the potential of the vaginal tract as an effective administration route for gene delivery. This review focuses on the main advantages and challenges of vaginal gene therapy, and on the most used nucleic acid delivery systems, including viral and non-viral vectors. Additionally, the advances in the application of vaginal gene therapy for the treatment and/or prevention of infectious diseases such as the human immunodeficiency virus (HIV), the human papillomavirus (HPV) or the herpes simplex virus (HSV) are presented.

Solid lipid nanoparticles as non-viral vector for the treatment of chronic hepatitis C by RNA interference.

RNA interference (RNAi) is a promising strategy to treat the chronic infection by hepatitis C virus (HCV). The objective of this work was to develop a non-viral vector based on solid lipid nanoparticles (SLN) and RNAi to inhibit the internal ribosome entry site (IRES) mechanism of the HCV. The vectors were prepared with SLN, protamine, hylauronic acid (HA) or dextran (DX), and a short-hairpin RNA expression plasmid targeted to the stem loop II of the 5' UTR (shRNA74). The particle size, surface charge, and capacity to bind, release and protect the shRNA74 against nucleases were evaluated. Cell uptake, silencing capacity and cell viability were evaluated in HepG2 cells. All the vectors presented particle size in the range of nanometers and positive surface charge, and they were able to protect the shRNA74 against DNase. An effective and rapid uptake into the cells was observed. Silencing capacity ranged from 3% to 67% depending on the presence of DX or HA in the vector, the shRNA74 to SLN ratio, and the shRNA74 dose. Vectors prepared with HA showed to be twice more effective than those prepared with DX. Differences in the intracellular trafficking may justify the higher efficacy of the HA-prepared vectors.

Treatment of ocular disorders by gene therapy.

Gene therapy to treat ocular disorders is still starting, and current therapies are primarily experimental, with most human clinical trials still in research state, although beginning to show encouraging results. Currently 33 clinical trials have been approved, are in progress, or have been completed. The most promising results have been obtained in clinical trials of ocular gene therapy for Leber Congenital Amaurosis, which have prompted the study of several ocular diseases that are good candidates to be treated with gene therapy: glaucoma, age-related macular degeneration, retinitis pigmentosa, or choroideremia. The success of gene therapy relies on the efficient delivery of the genetic material to target cells, achieving optimum long-term gene expression. Although viral vectors have been widely used, their potential risk associated mainly with immunogenicity and mutagenesis has promoted the design of non-viral vectors. In this review, the main administration routes and the most studied delivery systems, viral and non-viral, for ocular gene therapy are presented. The primary ocular disease candidates to be treated with gene therapy have been also reviewed, including the genetic basis and the most relevant preclinical and clinical studies.