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2 Non-viral Vectors for Gene Therapy Presently, more than 3400 genes have been associated with diseases [1], some of these pathologies are debilitating, mortal, and without any effective therapeutic options, and this number is expected to increase in the next decade as genomic studies advance. Gen...

2 Non-viral Vectors for Gene Therapy Presently, more than 3400 genes have been associated with diseases [1], some of these pathologies are debilitating, mortal, and without any effective therapeutic options, and this number is expected to increase in the next decade as genomic studies advance. Gene therapy is a therapeutic strategy that seeks to target the genes behind the disorders in order to cure patients. Thus, this approach holds the promise of establishing therapies that treat the causes, rather than the symptoms, by manipulating deficient genes, removing or silencing pathologic genes, or adding missing genes. However, these ambitious strategies have been hampered by several problems, such as the lack of safety and efficiency of some of the developed approaches, which can be explained by the very nature of most gene-­ manipulating tools. Naked DNA plasmids, for example, are rapidly degraded in biological fluids, they are unable to efficiently cross the cellular membranes and therefore reach the target cells and they activate the immune system, which is programmed to identify and eliminate vehicles containing foreign genetic information [2]. Thus, although a large number of preclinical and clinical studies have been performed using naked nucleic acids, the use of delivery vectors yields better results, namely because they protect the nucleic acids from nuclease degradation and increase intra- cellular delivery. Still, despite the big efforts made in the last decades with gene therapy strategies, very few gene therapy-based drugs or therapeutic products have reached the market, revealing that much still has to be done in the field of vectors for gene therapy. In fact, one of the major hurdles to the efficacious clinical application of gene therapy agents is the lack of efficient and safe delivery vehicles, which have to meet four basic criteria: (i) to protect the nucleic acids against degradation by nucleases; (ii) to deliver the nucleic acids into the cytoplasm or nucleus of the target cells; (iii) to be safe (i.e., trigger low levels of genotoxicity and immunogenicity); and (iv) to be economically viable (i.e., acceptable costs on a per patient basis). Gene therapy vectors are classically organized into two major groups, the non-viral and the viral vectors. The present chapter is focused on the non-viral vectors, which are subdivided into two categories: the physical and chemical methods. Physical methods (Fig. 2.1, Table 2.1) comprehend hydrodynamic delivery, microinjection, electroporation, nucleofection, sonoporation, gene gun, magnetofection, magnetoporation, microneedles, etc. Chemical vectors (Fig. 2.2, Table 2.2) include the polymeric-based systems, the lipid-based systems, metal nanoparticles, quantum dots, and graphene-based systems such as carbon nanotubes, silica nanoparticles, etc. © Springer Nature Switzerland AG 2020 C. Nóbrega et al., A Handbook of Gene and Cell Therapy, https://doi.org/10.1007/978-3-030-41333-0_2 23 2 24 Non-viral Vectors for Gene Therapy Fig. 2.1 Physical methods used for delivery of gene therapy tools. Different physical methods are available, namely, (i) hydrodynamic delivery that uses hydrostatic pressure enhancement to enlarge fenestrae and consequently tissue permeability; (ii) microinjection-mediated direct transfer of genetic material into the cell interior; (iii) electroporation and nucleofection, in which genetic material transfer is facilitated by electric pulses that cause transient pores in the cellular membrane and/or nuclear envelope; (iv) sonoporation, whereby delivery is triggered by transient cell membrane pores formed by ultrasounds; (v) ballistic delivery by accelerated nucleic acid-coated submicron metal particles; (vi) magnetofection and magnetoporation-­mediated gene delivery, based on the application of an external magnetic field promoting the guiding of the magnetic agent-containing vector to the target cells or promoting transient cellular pore formation, respectively; and (vii) microneedles used to deliver therapeutic agents across the skin. 2.1 Physical Methods 2.1.1 Hydrodynamic Delivery rise in venous or arterial pressure (depending on the injection site). This hydrostatic pressure increase will enlarge the fenestrae in the blood vessels (in the liver sinusoids, for example) and cause transient pores formation that result in the permeability enhancement of the tissue [3, 4]. This technique has been used with success in rodents; nevertheless, its application in humans is very limited. Regarding hydrodinamyc delivery to the liver in particular, the major concerns with are the transient elevation of hepatic The hydrodynamic derlivery technique is based on the enhancement of the pressure implemented in a tissue in order to increase the tissue permeability. Generally, this is performed through the rapid delivery of the nucleic acids in a high solution volume into the bloodstream. This rapid enhancement of the blood volume leads to a rapid 2.1 Physical Methods 25 Table 2.1 Main advantages and disadvantages of physical methods of gene therapy delivery Hydrodynamic delivery Microinjection Electroporation Nucleofection Ultrasound Ballistic gene delivery/gene gun Magnetofection Magnetoporation Microneedles Advantages Simple and efficient transfection Small amount of DNA required Efficient transfection and fast method Efficient in cells that are difficult to transfect and fast method Noninvasive strategy; can reach deeper organs and can be directed to specific areas Gene delivery to cells that are difficult to transfect Allows targeted cargo release and controlled release over time Used in cells that are difficult to transfect and requires no direct contact of electrodes Small amount of DNA required enzymes ­levels in the bloodstream, temporary cardiac dysfunction, high rise of venous pressure, and liver congestion caused by the injection of the high solution volume [4]. 2.1.2 Microinjection Microinjection consists in the direct transfer of genetic material into the cell interior. Generally, a glass micropipette with a submicron-sized diameter tip (less than 0.5 μm) is prefilled with the genetic material solution and is introduced into the target cell, under observation in a microscope. This technique may sound very straightforward; however it requires expensive equipment and extensive training of the operator, being technically very demanding. Additionally, (i) only a very small volume can be injected, otherwise additional stress can be triggered in the treated cells leading to intracellular environment and cell membrane disorganization; (ii) leakage of intracellular components at the injection site might occur, causing cell death; and (iii) the method is limited by cell size, being mostly applied to large cells, such as oocytes [5, 6]. Disadvantages Volumes required are too large for human application Difficult to perform and low performance Difficult to apply in vivo: difficulty to access target sites with electrodes and can cause tissue damage and inflammation Can cause high cell death and has a limited in vivo application Low efficiency of gene transfer and possible damaging of target cells Limited to a few-millimeter-­deep penetration and limited to superficial tissues and organs (muscles and skin) Requires specialized equipment and agglomeration of magnetofection reagents may occur after the magnetic field is removed Can cause cell death Difficult to perform and with a low performance 2.1.3 Electroporation Electroporation, also known as electropermeabilization and electrotransfer, is based on the application of a series of electrical pulses leading to a transient disruption of the cell membrane, allowing the passage of the genetic material into the cytosol of the cells. The exact mechanism underlying genetic material transfer into the cells is not known. However, it is known that, after employing an effective voltage (for in vivo application, the voltage is in the order of 200 V/cm), cell membrane becomes permeable, which is assumed to happen through the formation of hydrophilic membrane pores and consequent movement of genetic material through these pores as a local electrophoretic effect [3, 7]. The optimal duration and intensity of the electric pulses are different for different cell types and tissues to be transfected. For example, for skeletal muscle, better results come from protocols that first apply a pulse of high voltage, which will open membrane pores, followed by several low-voltage pulses, which will electrophorese the nucleic acids into the cells [3]. 26 Fig. 2.2 Chemical methods used for delivery of gene therapy tools. Chemical vectors include the polymeric-­ based systems, the lipid-based systems, metal nanoparticles, quantum dots, carbon nanotubes and silica-based nanoparticles. Polymer-based vectors are composed by a large number of repeated units bonded together that interact with the negatively charged nucleic acids, originating compact polyplexes that protect nucleic acids. Lipid-­ based systems comprise different delivery agents such as cationic lipoplexes formed through electrostatic interaction between the cationic lipids and negatively charged nucleic acids, and Smart/Trojan horse liposomes that can evade the immune system through the inclusion of hydrophilic polymers and that contain cell-targeting ligands to target their cargo to a specific cell population. High-­ density lipoprotein (HDL)-mimicking systems are HDL that are artificially prepared in order to efficiently deliver nucleic acids. Exosomes are membrane-based vesicles secreted by cells that can also be engineered to deliver 2 Non-viral Vectors for Gene Therapy nucleic acids. Inorganic materials such as metal nanoparticles, quantum dots, carbon nanotubes, and silica-based systems also promote efficient delivery of nucleic acids. Metal nanoparticles made up of noble metals such as gold and silver can be biofunctionalized and applied under specific conditions such as events triggered by light. Quantum dots are semiconductor crystals used as fluorescence probes to which it is possible to conjugate nucleic acids and a targeting ligand allowing the combination of the fluorophore properties and delivery functions in one system. Carbon nanotubes are made up of one or more sheets of graphene with cylindrical shape that form stable complexes with nucleic acids and to which it is possible to attach biomolecules to target them to a specific cell population and/or fluorescent probes to track them. Silica-­ based systems are silicon dioxide (SiO2, silica)-based nanoparticles that can be functionalized through the introduction of cationic components (polymers, lipids, etc.) that will complex the negatively charged nucleic acids. 2.1 Physical Methods 27 Table 2.2 Main advantages and disadvantages of polymer- and lipid-based systems of gene therapy delivery. Lipoplexes/ polyplexes Smart liposomes High-density lipoprotein (HDL)-mimicking systems Microvesicles-­ exosomes Advantages High transfection levels; possibility of choice of ideal lipids/polymers depending on administration route and targeted cells; large nucleic acid cargo capacity; easy to manufacture and low cost Functionalization leading to cell targeting and reduced interaction with plasmatic proteins Biodegradable, do not trigger immune reactions, and are less captured and removed from blood circulation by the reticuloendothelial system Biodegradable, do not trigger immune reactions This technique has been used with success for in vitro delivery of nucleic acids, as well as in some in vivo applications, such as for solid tumors in the skin and liver. The major limitations of this technique are the substantial cellular damage and consequent cytotoxicity that is triggered, as well as the limited in vivo application to local regions [7]. 2.1.4 Nucleofection Nucleofection technology is based on the same physical principles as electroporation; an electrical pulse is applied to the cells in order to open membrane pores through which the genetic material is internalized. However, in this method the exogenous material is directly delivered to the nucleus, being, therefore, a method used for cells that are more difficult to transfect. Moreover, the combination of the electric pulse with specific buffers significantly improves the outcomes. This methodology requires a low cell number, can be performed within minutes, and is very efficient in delivering the nucleic acids. On the other hand, in some cell types this method can trigger a high mortality [8–10]. 2.1.5 Disadvantages Interaction with plasmatic proteins can reduce half-life time and cause embolism More complex and expensive production More complex and expensive production Heterogeneity between batches related to size and composition (namely with respect to lipid, protein, and noncoding RNA content); poor pharmacokinetic profile in a systemic admnistration Ultrasound and Sonoporation Ultrasound is a mechanical vibration with a frequency above human audible range (20 kHz) that can be applied for nucleic acid delivery purposes. Ultrasound’s main action mechanism is the cavitation phenomenon. Cavitation is the process of formation and subsequent collapse of low-­ pressure voids (bubbles) driven by an acoustic field. These bubbles oscillate, grow, and implode releasing energy. The bubbles collapse causes sonoporation and local temperature and pressure rise. Sonoporation is the transient permeabilization (caused by pores formation) of the plasma membrane mediated by ultrasounds, which allows the transfer of nucleic acids into the cells [3, 11]. Microbubbles and some nucleation agents, such as ultrasound contrast agents, have been used to enhance the cavitation process, consequently increasing membrane permeabilization and leading to an improved gene transfer. Microbubbles are small bubbles (<10 μm) made of water-insoluble gas encapsulated in a biocompatible material that when irradiated by ultrasounds oscillate, generating enough fluid ­ flow (microflow) in the vicinity of the membranes to cause sonoporation [3, 11]. 2 28 Non-viral Vectors for Gene Therapy Ultrasound-mediated gene delivery is a noninvasive strategy that can reach deeper organs and can be directed to a specific area. netic field. A magnetic compound can be transported across the bloodstream and be concentrated in a particular organ of the body. Additionally, the cargo release can be controlled over time with the guidance of the magnetic 2.1.6 Ballistic Gene Delivery/ field. The major advantage of this strategy is the Gene Gun enhancement of the accumulation of nucleic acids at a specific site, which, for example, Ballistic gene delivery (or gene gun) is based on works well for solid tumor masses. However, in the bombardment of tissues by accelerated a situation that requires spreading of the theranucleic acid-coated particles. These submicron-­ peutic agent, such as metastatic, highly invasive, sized particles, usually gold particles, are first and infiltrative cancers, in which the cells are not covered with the nucleic acids to be delivered to accessible to a magnet, this technique has limthe cells and then they are accelerated in order to ited application [13, 14]. penetrate through the cell membrane, delivering The method of magnetoporation is also based their cargo into the cytosol [3, 5]. Several sys- on the use of magnetic fields to deliver nucleic tems for the particles acceleration are available; acids; in this case the field is used to promote the however, most of the devices use gas shock, pro- formation of transient pores in the cellular memduced either by a chemical explosion or a dis- branes, through which the nucleic acids cross charge of pressurized gas [5]. to the cell interior. This is possible because magThis strategy allows the delivery of genetic netic fields change the transmembrane potential material to a large surface and is also very use- of cells and, above a certain threshold, an ful in the case of cells that are difficult to trans- increased transmembrane potential can trigger fect, such as plant cells. In fact, the gene gun electroporation. The advantages of magnetoporawas originally developed to transform plants tion, as compared to electroporation, are as fol[12] that, given the rigidity of their cell wall, lows: (i) it requires no direct contact of electrodes are more difficult to penetrate at the cellular with the material to be permeabilized, being, level and to be genetically manipulated. therefore, noninvasive (“needle-less”); (ii) it is Although this is an interesting dellivery strat- easier and faster to apply; (iii) it is less expensive egy, the technology has the disadvantage of because it requires no disposables; and (iv) it has being limited to a few-­millimeter-­deep penetra- a bigger tissue penetration capacity, allowing tion and therefore is restricted to a more local- access to anatomical areas unreachable by elecized intervention [3]. troporation [14, 15]. 2.1.7 Magnetofection and Magnetoporation These delivery approaches are based on the application of an external magnetic field, which will promote the atomic dipole alignment of the material with the field, causing a magnetic moment within the material and its magnetization. Magnetofection, also known as magnetic transfection, is based on the incorporation of a magnetic agent in the vector, in order to deliver the nucleic acids under the influence of a mag- 2.1.8 Microneedles The use of microneedles to deliver therapeutic agents across the skin can be applied for DNA vaccine administration and to the delivery of nucleic acids in several skin pathologies, such as cutaneous cancers, wounds and hyperproliferative disorders. This delivery strategy provides means to overcome the skin barriers and to deliver the therapeutic agents directly into the dermal layers, allowing both localized and systemic delivery and avoiding the first-pass