Development and characterization of non-viral vectors based on cationic niosomes to address cystic fibrosis disease by gene therapy approach

246 p.Gene therapy is based on the delivery of exogenous genetic material into target cells to modulate theexpression of an altered genome in order to treat a specific disease. Lipid nanocarriers, such as niosomesbased on cationic lipids, non-ionic surfactants and ¿helper¿ components, are considered attractivecandidate for non-viral vectors due to their suitable biocompatibility and high versatility. The niosomechemical composition and their elaboration method influence the biophysical properties which have animpact on transfection efficiency and cytotoxicity. Indeed, compounds with specific properties have beenincluded to overcome some disadvantages of niosome formulations, such as chloroquine, which promotesendosomal escape. Gene therapy can be an excellent treatment for many disorders, in particular cysticfibrosis that is an autosomal monogenic recessive disease caused by different mutations in the cysticfibrosis conductance regulator (CFTR) gene. In this doctoral thesis, we focused on the development andin-depth biophysical and biological characterization of non-viral vectors based on cationic niosomes toface cystic fibrosis by gene therapy approach. The data obtained support that the inclusion of thechloroquine molecule in niosome formulations improves the biophysical properties of niosomes withenhanced transfection efficiencies and lower cytotoxicity. In addition, these niosomes are able to increasethe production of functional CFTR protein in cystic fibrosis cells. Furthermore, the development of athree-dimensional scaffold that better mimics the in vivo environment showed utility for evaluating newtreatments and different schedules of administration for cystic fibrosis

Gene Therapy for Cystic Fibrosis: Hurdles to Overcome for Successful Clinical Translation

Cystic fibrosis (CF) is a genetic disease that hampers the lung function. Despite that the main defective gene has been deeply characterized, some relevant concerns still need to be resolved before considering gene therapy as a realistic medical choice. One of the major issues that need to be strongly considered in order to succeed in the search for an effective gene therapy approach for CF is the design of the appropriate genetic material to be delivered. Other relevant factors to take into consideration include the design of safe and effective gene delivery systems, the biological barriers that need to be overcome in order to reach the nucleus of the target cells, and the problems related to the design of a drug formulation suitable for lung delivery purposes. Furthermore, some problems related to the commercialization of gene therapy products also need to be resolved. In this chapter, we discuss the up-to-date strategies to overcome such hurdles in order for gene therapy to become a routine treatment modality for CF

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.This work was supported by the Basque Country Government (Department of Education, University and Research, Consolidated Groups IT907-16) and by the Spanish Ministry of Science and Innovation (Grant PID2019-106199RB-C21). I.V.B. and M.S.R. thank the University of the Basque Country (UPV/EHU) for the granted postdoctoral fellowship (ESPDOC19/47) and the granted pre-doctoral fellowship (PIF17/79), respectively. Additional funding was provided by the CIBER of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), an initiative of the Carlos III Health Institute (ISCIII)

Clay Minerals as Bioink Ingredients for 3D Printing and 3D Bioprinting: Application in Tissue Engineering and Regenerative Medicine

The adaptation and progress of 3D printing technology toward 3D bioprinting (specifically adapted to biomedical purposes) has opened the door to a world of new opportunities and possibilities in tissue engineering and regenerative medicine. In this regard, 3D bioprinting allows for the production of tailor-made constructs and organs as well as the production of custom implants and medical devices. As it is a growing field of study, currently, the attention is heeded on the optimization and improvement of the mechanical and biological properties of the so-called bioinks/biomaterial inks. One of the strategies proposed is the use of inorganic ingredients (clays, hydroxyapatite, graphene, carbon nanotubes and other silicate nanoparticles). Clays have proven to be useful as rheological and mechanical reinforcement in a wide range of fields, from the building industry to pharmacy. Moreover, they are naturally occurring materials with recognized biocompatibility and bioactivity, revealing them as optimal candidates for this cutting-edge technology. This review deals with the use of clays (both natural and synthetic) for tissue engineering and regenerative medicine through 3D printing and bioprinting. Despite the limited number of studies, it is possible to conclude that clays play a fundamental role in the formulation and optimization of bioinks and biomaterial inks since they are able to improve their rheology and mechanical properties, thus improving printability and construct resistance. Additionally, they have also proven to be exceptionally functional ingredients (enhancing cellular proliferation, adhesion, differentiation and alignment), controlling biodegradation and carrying/releasing actives with tissue regeneration therapeutic activities.This research was funded by the BASQUE COUNTRY GOVERNMENT/EUSKO JAURLARITZA (Department of Education, University and Research, Consolidated Groups IT907-16). Authors S.R.-A. and M.S.-R. thank the BASQUE COUNTRY GOVERNMENT for the granted fellowship (PRE_2020_2_0143) and the UNIVERSITY OF THE BASQUE COUNTRY/EUSKAL HERRIKO UNIBERTSITATEA (UPV/EHU) for the granted pre-doctoral fellowship (PIF17/79), respectively

A Multicomponent Protocol for the Synthesis of Highly Functionalized γ-Lactam Derivatives and Their Applications as Antiproliferative Agents

An efficient synthetic methodology for the preparation of 3-amino 1,5-dihydro-2H-pyrrol-2-ones through a multicomponent reaction of amines, aldehydes, and pyruvate derivatives is reported. In addition, the densely substituted lactam substrates show in vitro cytotoxicity, inhibiting the growth of carcinoma human tumor cell lines HEK293 (human embryonic kidney), MCF7 (human breast adenocarcinoma), HTB81 (human prostate carcinoma), HeLa (human epithelioid cervix carcinoma), RKO (human colon epithelial carcinoma), SKOV3 (human ovarian carcinoma), and A549 (carcinomic human alveolar basal epithelial cell). Given the possibilities in the diversity of the substituents that offer the multicomponent synthetic methodology, an extensive structure-activity profile is presented. In addition, both enantiomers of phosphonate-derived γ-lactam have been synthesized and isolated and a study of the cytotoxic activity of the racemic substrate vs. its two enantiomers is also presented. Cell morphology analysis and flow cytometry assays indicate that the main pathway by which our compounds induce cytotoxicity is based on the activation of the intracellular apoptotic mechanism.Financial support by Ministerio de Economía, Industria y Competividad (MINECO) (RTI2018-101818-B-I00) and Gobierno Vasco (GV, IT 992-16) is gratefully acknowledged. The authors thank SGIker (UPV/EHU/ERDF, EU) for the technical and human support provided. X.d.C. and A.L.-F. thank the Basque Country Government for a predoctoral grant. I.V.-B. thanks the University of the Basque Country (UPV/EHU) for the granted postdoctoral fellowship (ESPDOC19/47). M.S.-R. thanks the University of the Basque Country (UPV/EHU) for the granted pre-doctoral fellowship (PIF17/79)

Assessment of Different Niosome Formulations for Optogenetic Applications: Morphological and Electrophysiological Effects

Gene therapy and optogenetics are becoming promising tools for treating several nervous system pathologies. Currently, most of these approaches use viral vectors to transport the genetic material inside the cells, but viruses present some potential risks, such as marked immunogenicity, insertional mutagenesis, and limited insert gene size. In this framework, non-viral nanoparticles, such as niosomes, are emerging as possible alternative tools to deliver genetic material, avoiding the aforementioned problems. To determine their suitability as vectors for optogenetic therapies in this work, we tested three different niosome formulations combined with three optogenetic plasmids in rat cortical neurons in vitro. All niosomes tested successfully expressed optogenetic channels, which were dependent on the ratio of niosome to plasmid, with higher concentrations yielding higher expression rates. However, we found changes in the dendritic morphology and electrophysiological properties of transfected cells, especially when we used higher concentrations of niosomes. Our results highlight the potential use of niosomes for optogenetic applications and suggest that special care must be taken to achieve an optimal balance of niosomes and nucleic acids to achieve the therapeutic effects envisioned by these technologies.This research was funded in part by grants RTI2018-098969-B-I00, PRE2019-087693, DTS19/00175, and PDC2022-133952-100 from the Spanish “Ministerio de Ciencia, Innovación y Universidades” and by the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 899287 (NeuraViPeR)

Sphingolipid extracts enhance gene delivery of cationic lipid vesicles into retina and brain

[EN]The aim was to evaluate relevant biophysic processes related to the physicochemical features and gene transfection mechanism when sphingolipids are incorporated into a cationic niosome formulation for non-viral gene delivery to central nervous system. For that, two formulations named niosphingosomes and niosomes devoid of sphingolipid extracts, as control, were developed by the oil-in water emulsion technique. Both formulations and the corresponding complexes, obtained upon the addition of the reporter EGFP plasmid, were physicochemically and biologically characterized and evaluated. Compared to niosomes, niosphingosomes, and the corresponding complexes decreased particle size and increased superficial charge. Although there were not significant differences in the cellular uptake, cell viability and transfection efficiency increased when human retinal pigment epithelial (ARPE-19) cells were exposed to niosphingoplexes. Endocytosis via caveolae decreased in the case of niosphingoplexes, which showed higher co-localization with lysosomal compartment, and endosomal escape properties. Moreover, niosphingoplexes transfected not only primary central nervous system cells, but also different cells in mouse retina, depending on the administration route, and brain cortex. These preliminary results suggest that niosphingosomes represent a promising non-viral vector formulation purposed for the treatment of both retinal and brain diseases by gene therapy approach.This work was supported by the Basque Country Government (Department of Education, University and Research, Consolidated Groups IT907-16) . Additional funding was provided by the CIBER of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) , an initiative of the Carlos III Health Institute (ISCIII) . I.V.B. and M.S.R. thank the University of the Basque Country (UPV/EHU) for the granted postdoctoral fellowship (ESPDOC19/47) and the granted pre-doctoral fellowship (PIF17/79) , respectively. Authors wish to thank the intel-lectual and technical assistance from the ICTS "NANBIOSIS," more specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/EHU) . Technical and human sup-port provided by SGIKER (UPV/EHU) is also gratefully acknowledged

Niosome-Based Approach for In Situ Gene Delivery to Retina and Brain Cortex as Immune-Privileged Tissues

Non-viral vectors have emerged as a promising alternative to viral gene delivery systems due to their safer profile. Among non-viral vectors, recently, niosomes have shown favorable properties for gene delivery, including low toxicity, high stability, and easy production. The three main components of niosome formulations include a cationic lipid that is responsible for the electrostatic interactions with the negatively charged genetic material, a non-ionic surfactant that enhances the long-term stability of the niosome, and a helper component that can be added to improve its physicochemical properties and biological performance. This review is aimed at providing recent information about niosome-based non-viral vectors for gene delivery purposes. Specially, we will discuss the composition, preparation methods, physicochemical properties, and biological evaluation of niosomes and corresponding nioplexes that result from the addition of the genetic material onto their cationic surface. Next, we will focus on the in situ application of such niosomes to deliver the genetic material into immune-privileged tissues such as the brain cortex and the retina. Finally, as future perspectives, non-invasive administration routes and different targeting strategies will be discussed.This work was supported by the Basque Country Government (Department of Education, University and Research, pre-doctoral grant PRE_2016_2_0302 and Consolidated Groups IT907-16). Additional funding was provided by the University of Basque Country UPV/EHU (predoctoral grant PIF17/19), the CIBER of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), and initiative of the Carlos III Health Institute (ISCIII)

Multicomponent Synthesis of Unsaturated γ-Lactam Derivatives. Applications as Antiproliferative Agents through the Bioisosterism Approach: Carbonyl vs. Phosphoryl Group

We report efficient synthetic methodologies for the preparation of 3-amino and 3-hydroxy 3-pyrrolin-2-ones (unsaturated γ-lactams) through a multicomponent reaction of amines, aldehydes and acetylene or pyruvate derivatives. The densely substituted γ-lactam substrates show in vitro cytotoxicity, inhibiting the growth of the carcinoma human tumor cell lines RKO (human colon epithelial carcinoma), SKOV3 (human ovarian carcinoma) and A549 (carcinomic human alveolar basal epithelial cell). In view of the possibilities for the diversity of the substituents that offer a multicomponent, synthetic methodology, an extensive structure–activity profile is presented. In addition, the bioisosteric replacement of the flat ester group by a tetrahedral phosphonate or phosphine oxide moiety in γ-lactam substrates leads to increased growth inhibition activity. Cell morphology analysis and flow cytometry assays indicate that the main pathway by which our compounds induce cytotoxicity is based on the activation of the intracellular apoptotic mechanism.Financial support by Ministerio de Economía, Industria y Competividad (MINECO) (RTI2018-101818-B-I00) and Gobierno Vasco (GV, IT 992-16) is gratefully acknowledged. X.d.C. and A.L.-F. thank the Basque Country Government for a predoctoral grant. I.V.-B. thanks the University of the Basque Country (UPV/EHU) for a postdoctoral fellowship (ESPDOC19/47). M.S.-R. thanks the University of the Basque Country (UPV/EHU) for a pre-doctoral fellowship (PIF17/79)

Correlation between Biophysical Properties of Niosomes Elaborated with Chloroquine and Different Tensioactives and Their Transfection Efficiency

Lipid nanocarriers, such as niosomes, are considered attractive candidates for non-viral gene delivery due to their suitable biocompatibility and high versatility. In this work, we studied the influence of incorporating chloroquine in niosomes biophysical performance, as well as the effect of non-ionic surfactant composition and protocol of incorporation in their biophysical performance. An exhaustive comparative evaluation of three niosome formulations differing in these parameters was performed, which included the analysis of their thermal stability, rheological behavior, mean particle size, dispersity, zeta potential, morphology, membrane packing capacity, affinity to bind DNA, ability to release and protect the genetic material, buffering capacity and ability to escape from artificially synthesized lysosomes. Finally, in vitro biological studies were, also, performed in order to determine the compatibility of the formulations with biological systems, their transfection efficiency and transgene expression. Results revealed that the incorporation of chloroquine in niosome formulations improved their biophysical properties and the transfection efficiency, while the substitution of one of the non-ionic surfactants and the phase of addition resulted in less biophysical variations. Of note, the present work provides several biophysical parameters and characterization strategies that could be used as gold standard for gene therapy nanosystems evaluation