• Título:  DEVELOPMENT AND EVALUATION OF NON-VIRAL GENE DELIVERY VECTORS AND THEIR COMBINATION WITH HYDROGEL SCAFFOLD TECHNOLOGY
• Autor:  VILLATE BEITIA, ANE ILIA
• Universidad:  Universidad del País Vasco/Euskal Herriko Unibertsitatea
• Departamento:  Farmacia y ciencias de los alimentos
• Fecha de Lectura:  26/10/2018
• Mención Europeo / Mención Internacional: concedido
• Programa de doctorado:  Programa de Doctorado en Investigación y Evaluación de Medicamentos. Aplicación de la Tecnología Farmacéutica al Desarrollo de Terapias Avanzadas por la Universidad del País Vasco/Euskal Herriko Unibertsitatea
• Dirección: 
  • PEDRAZ MUÑOZ, JOSÉ LUIS (DIRECTOR)
  • ZARATE SESMA, JON (DIRECTOR)
• Tribunal: 
  • ALMEIDA, ANTONIO JOSE (PRESIDENTE)
  • PURAS OCHOA, GUSTAVO (SECRETARIO)
  • SOTO SÁNCHEZ, CRISTINA (VOCAL)
  • MARTINEZ DE PANCORBO GOMEZ, MARIA DE LOS ANGELES (VOCAL)
  • MONTOLIU JOSE, LLUIS (VOCAL)
• Descriptores: 
  • COMPOSICION DE FARMACOS
• Localización:  REPOSITORIO INSTITUCIONAL E.H.U./U.P.V.
• http://hdl.handle.net/10810/32566
• Resumen:
  Nanotechnology-based non-viral vectors have emerged as promising alternatives to viruses to carry genetic material into target cells due to their ability to overcome many limitations of viral vectors. Although very efficient, viruses present low carrying capacity, expensive and complex production and, most importantly, safety issues related to immunogenic responses and even oncogenesis when randomly integrated in the host genome. In this regard, non-viral vectors present lower immunogenicity, higher nucleic acid packing capacity and ease of fabrication compared to their viral counterparts. In addition, continuous advances in the fields of material science and nano-engineering as well as the diversity of available nanosized material allows the design of multifunctional vectors specifically tailored for different applications. Although most conventional gene therapy strategies based on gene addition use viral vectors, nowadays non-viral vectors are predominant in new investigations based on gene editing due to their favorable characteristics. To date, a wide variety of nanosized non-viral gene delivery vectors have been developed, including cationic lipids, polymers and magnetic nanoparticles. These molecules can be nanoengineered to pass over several extracellular and intracellular barriers and to transport therapeutic genes into specific organs or cell types, including cells in the central nervous system (CNS), one of the most challenging organs for both viral and non-viral gene delivery systems.Among the wide plethora of non-viral vectors, recently emerged niosomes are biocompatible, synthetic, non-ionic surfactant vesicles with a closed bilayer structure. Niosomes have been demonstrated to be effective for retinal gene transfer, an important target organ in many gene therapy strategies due to the existence of several well characterized monogenic retinal disorders. Niosome formulations are based on three principal components -cationic lipids, helper lipids, and non-ionic surfactants. The global chemical properties of these components influence on the physicochemical characteristics of niosomes, such as size, surface charge and morphology, which in turn determine their ability to enter the cells, follow a particular endocytic pathway, deliver the DNA cargo into the nucleus and, therefore, their transfection efficiency. The roles of cationic lipids and helper lipids in the transfection process and efficiency mediated by niosomes have been thoroughly studied in rat retina and brain. However, the influence of the non-ionic surfactant component also needs to be well characterized in order to optimize the design of niosome formulations for retinal gene delivery and be able to achieve persistent and high levels of transgene expression, necessary for biomedical applications in retinal disorders.Successful gene transfer not only depends on the carrier, but the properties of the vectored genetic material are also important. Until recently, most efforts have focused on improving nucleic acid delivery strategies either by modifying non-viral vectors or by employing physical methods such as electroporation. Nevertheless, the enhancement of transfection efficiency can also be achieved by modifying the composition and conformation of the genetic material in order to improve its bioavailability, biocompatibility, durability and safety. In this regard,Development and evaluation of non-viral gene delivery vectors and their combination with hydrogel scaffold technology.2minimized expression units devoid of bacterial backbone and that only contain the gene of interest and regulatory sequences such as minicircle (MC) DNA vectors emerge as an attractive alternative to conventional plasmid DNA vectors. In fact, MC DNA vectors offer several advantages over their larger parental plasmid DNA vectors, including enhanced transfection efficiency with sustained transgene expression as well as improved immunocompatibility and safety profiles. High levels of transgene expression mediated by MC DNA vectors have been demonstrated in a variety of organ systems in vivo including the liver, heart and skeletal muscle, but MC DNA technology has not yet been assessed for retinal gene delivery in complex with niosome formulations.Finally, in addition to the optimization of the carrier and the genetic material, a new trend in non-viral gene therapy is to explore novel combined strategies between delivery systems and tissue-engineered scaffolds or matrix design. In fact, these combined approaches may offer relevant advantages such as enhanced stability and reduced toxicity. Moreover, complementing gene transfer with matrix design would allow for an effective, targeted and local DNA delivery, which would promote the applicability of gene therapy in many therapeutic fields such as tissue regeneration. Hyaluronic acid (HA) hydrogels have been widely studied for their biocompatibility as well as their ability to incorporate a wide variety of molecules, including nucleic acids. In addition, polymeric non-viral vectors based poly(ethylene imine) (PEI) have been encapsulated into HA hydrogels with successful local transgene expression, but the biomedical application of PEI derivatives is often restricted due to immunogenicity and cytotoxicity issues. In this regard, niosomes offer several advantages due to their high compatibility with biological systems and low toxicity, but the combination between niosome gene delivery systems and HA hydrogel scaffolds has not yet been explored.In view of these considerations, the main objective of the present work was to evaluate key factors that determine the transfection process and efficiency of non-viral vectors, including the composition and physicochemical properties of non-viral vectors, the composition and conformation of the genetic material and the combination of delivery systems with other technologies such as matrix design. In order to accomplish that purpose, four experimental chapters were designed and developed (Figure 1).Figure 1. Summary of the four experimental chapters developed in the doctoral thesis.Development and evaluation of non-viral gene delivery vectors and their combination with hydrogel scaffold technology.3In the first chapter, the transfection efficiency of three different non-viral vectors based on cationic lipids, polymers and magnetic nanoparticles was evaluated and compared. In this chapter, the genetic material employed for transfection corresponded to a therapeutic plasmid coding for the vascular endothelial growth factor (VEGF) and the target organ was the central nervous system (CNS). From this first chapter, we learned that nanoparticles based on lipoplexes, polyplexes and magnetoplexes showed suitable physicochemical characteristics for gene delivery, magnetoplexes showing the highest ability among the three formulations to transfect CNS cells. In addition, the VEGF protein produced by primary neuronal cell cultures transfected with magnetoplexes maintained its bioactivity.In the second chapter, the study focused on niosome based non-viral vectors, specifically on the role of the non-ionic tensioactive component of niosome formulations in retinal transfection processes both in vitro and in vivo. From this work, we learned that polysorbate 20 non-ionic surfactant enhanced gene delivery mediated by niosome formulations into retinal cells in vitro and in vivo. Also, it was shown that nioplexes containing polysorbate 20 entered retinal ARPE-19 cells via caveolae mediated endocytosis in vitro. Moreover, regarding in vivo studies in rat retina, subretinal administration of nioplexes based on polysorbate 20 showed transfection in different layers of the retina, including the retinal pigment epithelium, while intravitreal administration of this formulation showed transfection predominantly in the ganglion cell layer of the retina.The third chapter was designed in order to understand the influence of the genetic material in retinal transfection processes mediated by niosome formulations both in vitro and in vivo. In this study, we learned that niosome formulations containing different DNA constructs presented similar physicochemical characteristics suitable for gene delivery but differed in transfection efficiency in retinal cells. It was shown that MC DNA technology enhanced retinal transfection in vitro as well as in vivo in rat retina. Additionally, stability studies of niosome formulations demonstrated that after 30 days of storage, transfection efficiency of these formulations decreased, specially if stored at room temperature. In vivo studies carried in this chapter also showed that transfection efficiency in different retinal layer depended on the administration route, being the subretinal administration more suitable to transfect the retinal pigment epithelium cell layer and the intravitreal administration better to transfect the ganglion cell layer of the retina. These results are consistent with the previous chapter.Finally, in the fourth chapter of this doctoral thesis, we explored new combinations between niosome-based gene delivery systems and hyaluronic acid hydrogel scaffolds for 3D transfection and local gene delivery. This study was developed during the international predoctoral stay in the Molecular Engineering Department of the University of California in Los Angeles (UCLA). The research developed during that period demonstrated that niosome formulations composed of 2,3-di(tetradecyloxy)propan-1-amine, Poloxamer 407, polysorbate 80 and chloroquine diphosphate salt can be successfully incorporated into hyaluronic acid hydrogels for non-viral gene delivery. Hyaluronic acid hydrogel scaffolds loaded with nioplexes presented suitable mechanical properties, little or no particle aggregation, allowed for extensive cell spreading and were able to efficiently transfect encapsulated mMSCs in 3D cultures.Development and evaluation of non-viral gene delivery vectors and their combination with hydrogel scaffold technology.4As a whole, this doctoral thesis describes and explores key factors that determine the transfection efficiency of non-viral vectors and that need to be taken into account when developing efficient non-viral gene delivery systems. Those key aspects include the composition and physicochemical properties of non-viral vectors, the composition and conformation of the genetic material and the combination of delivery systems with other technologies such as matrix design. We believe that the concept of a unique universal non-viral vector is nowadays obsolete, and that future non-viral gene delivery platforms will be based on multifunctional vectors specifically tailored for different applications. Although non-viral vectors are still far from clinical practice, reasonable hope suggests that next generation delivery systems for gene addition and, most importantly, gene editing strategies may be based non-viral vector systems, which, ideally, would be suitable for administration via non-invasive routes.