**1. Introduction**

380 Non-Viral Gene Therapy

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#### **1.1 Chapter objectives**

Gene therapy is the process by which a foreign, corrective (or missing) gene is inserted toward biological tissues or cells aiming to alleviate symptoms or prevent disorders. Several clinical trials have demonstrated gene therapy as a promising option to treat diseases. However, therapeutic biological limitations (such as adverse immune responses of the body to incoming gene delivery systems) coupled with a poor understanding of the physicochemical motifs involved in the DNA compaction and delivery (transfection) processes have resulted in non-100% effective protocols. Aiming to contribute to a better understanding of the different physicochemical aspects of gene therapy, our group has committed for some years now to the physical chemistry characterization of the DNA compaction and transfection mediated by different kinds of compacting agents (vectors).

In this chapter, we present an overview of the results we have obtained during the last three years regarding the DNA compaction and transfection mediated by cationic-liposomes and polymers (polycations). Two families of polycations, Chitosan and Poly (diallyldimethylammonium chloride) (pDADMAC), and one cationic lipid formulation extensively used worldwide in transfection assays, Metafectene® Pro (MEP), are studied as DNA vectors and compared with other systems already published. In particular, by varying the solution pH and polycation characteristics (chemical composition and molecular weight), we assess the influence of polycation-charge density (i.e., the mole fraction of the ionized groups along the polymer chain) and -valence (i.e., the total charge per polymer chain) on different parameters of the complexes formed that are important for gene therapy. The studied parameters include i) the hydrodynamic radius, RH, ii) the stability with time, iii) the vector to DNA ratio at which complexation takes place iv) the ζ-potential, v) the

Polycation-Mediated Gene Delivery: The Physicochemical Aspects Governing the Process 383

(Felgner et al., 1987). Other non-viral vectors subsequently studied include surfactants, proteins (particularly histones), multivalent ions, nanoparticles, and polycations, all of which (also) form electrostatically driven DNA complexes (Gonzalez-Perez & Dias, 2009). In the following sections we explore representative gene delivery systems employing

Polycations and cationic liposomes are the non-viral vectors most commonly studied for gene therapy due to the outstanding characteristics they present. In addition to the potential safety benefits, they offer, for instance, a great structural and chemical versatility for manipulating their physicochemical properties, vector stability upon storage and reconstitution, and a larger gene capacity to transfer DNA as compared to their viral and non-viral counterparts (Dias et al., 2002; Midoux et al., 2009; Tros de Ilarduya et al., 2010). As mentioned before, cationic liposomes were the first class of non-viral vectors showing satisfactory transfection efficiencies. In the first work reporting on lipofection (the lipidmediated DNA transfection process), Felgner and coworkers employed N-[1-(2,3 dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), a synthetic cationic lipid, to transfect plasmid DNA to different cells lines in culture (Felgner et al., 1987). Major advantages of utilizing DOTMA containing liposomes were that DNA entrapment inside the lipoplexes was found to be of a 100%. Also, as suggested by fluorescence microscopy data, DOTMA demonstrated to facilitate fusion of the complexes with the plasma membrane of the studied culture cells, resulting in DNA transfer rates from 5- to >100-fold more effective (depending on the transfected cell line) than previous procedures such as the calcium phosphate or the DEAE-dextran transfection techniques (Kucherlapati & Skoultchi, 1984). However, a major drawback was that these liposomes were found to be cytotoxic (Felgner et al., 1987). Following the trail of this pioneering work, other cationic lipids and surfactants have also been tested over the years (Simberg et al., 2004). Unfortunately, the general perspective is that excessive positive charges, facilitating the electrostatic interactions with negatively charged DNA, also promote cytotoxicity. As a result, zwitterionic lipids such as dioleoylphosphatidylethanolamine (DOPE) and cholesterol are

Polycations (i.e., positively charged polyelectrolytes) are macromolecules attractive to gene therapy for various reasons. Firstly, provided the high charge density they bear, they are considered as the most efficient nucleic acid-condensing agents. Different to other kinds of non-viral vectors like trivalent ions and cationic surfactants, interacting with a few consecutive DNA monomers (bases) (Sarraguca & Pais, 2006), polycations interact with DNA bases that are significantly far apart, promoting bridging between different sites in the

Secondly, because of the strong DNA-polycation interactions, DNA-polycation complexes (polyplexes) are specially effective in what DNA charge masking and extracellular protection concerns (De Smedt et al., 2000). And thirdly, given that they can be functionalized, copolymerized or structurally modified, polycation constructs can be tailored to improve cell-specific therapeutic efficacy with reduced side effects (Ke & Young, 2010). Polycations most frequently studied as gene carriers include poly(L-lysine) (PLL) (G. Y. Wu & C. H. Wu, 1987), polyethylenimine (PEI) (Boussif et al., 1995), chitosan (Mumper et al., 1995), poly(β-amino ester)s (Lynn & Langer, 2000), and poly(amido amine) (PAMAM) dendrimers (Haensler & Szoka, 1993). Generally speaking, the basicity

polycations and cationic liposomes as gene carriers.

**1.3 Cationic-liposomes and -polymers for gene therapy** 

nowadays commonly implemented (Tros de Ilarduya et al., 2010).

DNA chain or between different DNA chains (Dias et al., 2003).

binding energetics, vi) the morphology, and vii) the transfection efficiency. The physicochemical characterization was carried out by means of different experimental techniques including dynamic and static light scattering (DLS and SLS), electrophoretic mobility, isothermal titration calorimetry (ITC), transmission electron- and atomic force microscopy (TEM and AFM), and conductometry at 25 ºC. Transfection experiments were conducted at standard culture conditions and evaluated by means of the β-galactosidase (βgal) and luciferase assays at 25 ºC. Outstanding results concerning the electrochemical and energetic features of the complexes with higher transfection efficiencies are fully discussed.

#### **1.2 Gene therapy**

The possibility to treat diseases by the insertion of genes into human cells and tissues has proposed gene therapy as the therapy of the 21st century (Verma & Somia, 1997). With the first clinical trial reported in the early 1990s (Anderson, 1990), protocols for several, diverse disorders have been conducted and promising results have been obtained (O'Connor & Crystal, 2006); however, a single protocol suitable to be applied as a routinely means to treat any particular disease is far to be achieved.

In practice, the entrance of naked, exogenous DNA to the cell nucleus results problematic due to different extra and intracellular barriers. On the one hand, systemic circulation of DNA is hindered by nuclease degradation (Nguyen et al., 2009). On the other hand, the electrolytic nature of DNA gives rise to electrostatic repulsions as DNA approaches to cells provided that both DNA and cell membranes are negatively charged (due to the phosphate groups distributed by the outside of the polymer helices and the several proteoglycans constituting the cell membranes) (Tros de Ilarduya et al., 2010). Also, once inside cells, steric restrictions are expected to hamper the DNA transportation to the cell nucleus given that mobility of free DNA based on diffusion in the cytoplasm is negligible (Dowty et al., 1995), possibly due to cytoskeletal elements within the cytoplasm that function as molecular sieves and prevent the diffusion of large molecules (Lubyphelps et al., 1987). Thus, for exogenous DNA to be properly transferred to living cells (and tissues), all these extra and intracellular barriers must be circumvented.

Current gene transfer protocols rely on the use of natural and synthetic DNA complexing agents, referred to as vectors or gene carriers, to compact, protect, and provoke a charge inversion of DNA, surpassing, by this way, the previously cited biological barriers. Vectors are either viral or non-viral. Viral delivery, also known as transduction, involves the packaging of DNA (or in some cases RNA) into a virus particle (Mancheño-Corvo & Martín-Duque, 2006). This procedure is, by far, the most effective one considering the high transfection efficiencies it renders; however, fundamental problems associated with viral vectors, including toxicity and immunogenicity, among others, have encouraged the investigation of safer gene delivery alternatives such as non-viral vectors (Verma & Somia, 1997).

Compared to viruses, non-viral transfection vectors possess many important advantages such as safety, versatility, ease of preparation, and, in some cases, the possibility to transfect DNA fragments of unlimited sizes (Orth et al., 2008). The first approaches using non-viral vectors as gene carriers come from the late 1980s when Felgner and coworkers started to try with cationic liposomes (Felgner et al., 1987). Cationic liposome-DNA complexes, also referred to as lipoplexes, form spontaneously after electrostatic interactions between the positively charged liposomes and the negatively charged DNA, producing physically stable formulations suitable to transfect relatively high amounts of plasmid DNA to cells in culture (Felgner et al., 1987). Other non-viral vectors subsequently studied include surfactants, proteins (particularly histones), multivalent ions, nanoparticles, and polycations, all of which (also) form electrostatically driven DNA complexes (Gonzalez-Perez & Dias, 2009). In the following sections we explore representative gene delivery systems employing polycations and cationic liposomes as gene carriers.
