**2.6 Delivery methods**

Different methods for enhancing naked DNA vaccine delivery into host cells have been studied. The best investigated strategies are based on chemical or physical devices aimed at facilitating the DNA entry into recipient cells.

#### **2.6.1 Microparticles**

Microparticle-based methods operate a DNA condensation and complexation in particles (O'Hagan et al. 2004). The encapsulation of plasmid DNA into micro- or nanospheres can provide protection from the environment prior to delivery and aid in targeting to a specific cell type for efficient delivery.

The major advantage of particulate delivery is that synthetic microparticles have excellent potential for targeting cells of the immune system stimulating antigen uptake. It has been demonstrated that particles of about 1-10 μm in diameter are preferred for their size that is readily phagocytosed by dendritic cells and other antigen-presenting cells. They are readily internalised by phagocytic cells of the immune system, leading to an enhanced antigen presentation to the immune effector cells. Furthermore, microparticulates appear to improve delivery of DNA to APCs by facilitating trafficking to the local lymphoid tissue via the afferent lymph and antigen uptake by dendritic cells (Denis-Mize et al. 2000; Denis-Mize et al. 2003; Dupuis et al. 2000). Moreover, antigen and adjuvant molecules can be delivered to the same cell at the same time being entrapped together in biodegradable microparticles such as poly-lactide-co-glycolide (PLG) or chitosan, or complexed with non-ionic block copolymers or polycations such as polyethyleneimine. Microparticulate adjuvants are currently tested in some clinical trials against human immunodeficiency virus (HIV), hepatitis B virus (HBV) and influenza (Fuller et al. 2006). DNA entrapment or encapsulation into biodegradable microspheres for DNA vaccine delivery has been illustrated in patent WO0203961 (Johnson 2003).

#### **2.6.2 Cationic lipids/liposomes**

182 Non-Viral Gene Therapy

Aluminium salt adjuvants (aluminium hydroxide, aluminium phosphate) are generally used in combination with protein antigens as they form a precipitated or adsorbed vaccine. An efficient, safe and well tolerated adjuvant in humans is aluminium hydroxide (Alum). It has been approved for clinical use. Although traditionally thought to function primarily by forming a long-lasting depot for antigen and by promoting their uptake by APCs, it is now clear that innate immune stimulation plays a primary role in the adjuvant activity of alum (Lambrecht et al. 2009). Aluminium hydroxide is used primarily to enhance antibody production and does not utilize TLR for its function in vivo (Gavin et al. 2006).This adjuvant induces a Th-2 biased immune response in mice whereas in humans it stimulates also a Th-1 type immunity. *In vitro* studies demonstrated that Alum can activate the inflammosome

Recently, aluminium hydroxide has been also shown to work well in DNA vaccination in pre-clinical models (Kenney and Edelman 2003). However, the enhancing effect of aluminium hydroxide on the immune response elicited by DNA vaccines, is not related to the levels of antigen expression. Rather, it seems to affect antigen after *in vivo* expression, suggesting the adjuvanticity of this substance is strictly related to the antigen delivery

Different methods for enhancing naked DNA vaccine delivery into host cells have been studied. The best investigated strategies are based on chemical or physical devices aimed at

Microparticle-based methods operate a DNA condensation and complexation in particles (O'Hagan et al. 2004). The encapsulation of plasmid DNA into micro- or nanospheres can provide protection from the environment prior to delivery and aid in targeting to a specific

The major advantage of particulate delivery is that synthetic microparticles have excellent potential for targeting cells of the immune system stimulating antigen uptake. It has been demonstrated that particles of about 1-10 μm in diameter are preferred for their size that is readily phagocytosed by dendritic cells and other antigen-presenting cells. They are readily internalised by phagocytic cells of the immune system, leading to an enhanced antigen presentation to the immune effector cells. Furthermore, microparticulates appear to improve delivery of DNA to APCs by facilitating trafficking to the local lymphoid tissue via the afferent lymph and antigen uptake by dendritic cells (Denis-Mize et al. 2000; Denis-Mize et al. 2003; Dupuis et al. 2000). Moreover, antigen and adjuvant molecules can be delivered to the same cell at the same time being entrapped together in biodegradable microparticles such as poly-lactide-co-glycolide (PLG) or chitosan, or complexed with non-ionic block copolymers or polycations such as polyethyleneimine. Microparticulate adjuvants are currently tested in some clinical trials against human immunodeficiency virus (HIV), hepatitis B virus (HBV) and influenza (Fuller et al. 2006). DNA entrapment or encapsulation into biodegradable microspheres for DNA vaccine delivery has been illustrated in patent

**2.5.3 Aluminium-based compounds** 

pathway to produce IL1-β (Li et al. 2007).

facilitating the DNA entry into recipient cells.

mechanism (Ulmer et al. 1999).

**2.6 Delivery methods** 

**2.6.1 Microparticles** 

cell type for efficient delivery.

WO0203961 (Johnson 2003).

Cationic Solid Lipid Nanoparticles (SLNs) have been recently proposed as alternative carriers for DNA delivery, due to many technological advantages such as large-scale production from substances generally recognized as safe, good storage stability and possibility of steam sterilization and lyophilisation. Cationic lipids are amphiphilic molecules composed of one or two fatty acid side chains (acyl) or alkyl, a linker and a hydrophilic amino group. The hydrophobic part can be cholesterol-derived moieties. In aqueous media, cationic lipids are assembled into a bilayer vesicular-like structure (liposomes). Liposomes/DNA complex is usually termed a lipoplex (Bolhassani et al. 2011). The future success of cationic SLNs for administration of genetic material will depend on their ability to efficiently cross the physiological barriers, selectively targeting a specific cell type in vivo and expressing therapeutic genes (Bondi and Craparo 2010).

#### **2.6.3 Biolistic particle delivery**

In order to address accelerating micro-projectiles into intact cells or tissues, is generally used a biolistic apparatus described in patent US6004287 (Loomis 1999). Application of this strategy to DNA vaccines resulted in the invention of a new DNA delivery technology that made it possible to move naked DNA plasmid into target cells on an accelerated particle carrier. This specific delivery system is based on the use of the gene gun device that, under pressurized helium, is capable of delivering plasmid DNA-coated gold beads to the epidermal layer of skin as described in patent US6436709 (Lin 2002). Because the DNA carrier is introduced directly into the skin cells, delivery of plasmid DNA vaccines using this strategy reduces the amount of DNA needed to induce immune responses. Robust immunogenicity has been shown in many different preclinical models and in clinical trials predominantly for infectious diseases (Fuller et al. 2006). In contrast to intramuscular or intradermal injection by needle, the gene gun delivery system releases plasmid DNA directly into the cells of the epidermis (Yang et al. 1990). Intradermal injection is becoming increasingly popular, as the dense network of antigen-presenting cells in the skin, absent in muscle, provides a favourable environment for induction of antigen uptake. This network of Langerhans cells (LCs) can help in the priming of both cellular and humoural immune responses. Importantly, direct transfection of Langerhans cells is carried out with very small doses of plasmid DNA (i.e. 1-10 μg), suggesting that minimum amounts of vector are required to induce the immune response. The advantage of using low doses of plasmid DNA is particularly attractive for prophylactic vaccines against infectious diseases, where a simple and rapid delivery is the main pre-requisite. Gene gun delivery has recently been used with success in a trial against the influenza virus, inducing sero-protective levels of antibody and it has been used in trials against HBV and HIV infections (Fuller et al. 2006). A further implementation of the biolistic delivery was obtained also by creating improved injection device suitable for application in human tissues. Patent US6730663 describes a flexible multi-needle injector device with a wide surface area as well as a modified injector device to be used for injection through an endoscopic device. Such a method leads to a deep injection of DNA within tissues (Hennighausen 2004).

#### **2.6.4 Electropermeabilization**

The use of electric pulses as a safe tool to deliver therapeutic molecules to tissues and organs has been rapidly developed over the last decade. This technology leads to a transient increase in the permeability of cell membranes when exposed to electric field pulses. This

DNA Vaccination by Electrogene Transfer 185

and Th-1 biased immune responses (Gronevik et al. 2005). Another study demonstrated that a single i.m. DNA vaccination in combination with EP enhanced significantly the onset and the duration of the primary antibody response affecting immune memory (Tsang et al. 2007). The use of EP for delivering a DNA vaccine encoding anthrax toxin protective agent has been demonstrated able in a rapid induction of antibodies against the antigen in 2 weeks following a single immunization in several experimental animals (Luxembourg et al. 2008). Despite these evidences, details on possible mechanisms responsible for the positive effect of EP on the immune response to DNA vaccines were not completely characterised (Escoffre et

Recently EP has been reported as crucial event through which, inducing transient morphological changes and a local moderate damage in the treated muscle, is possible to generate an early production of endogenous cytokines responsible for signalling danger at the local level. The activation of a danger pro-inflammatory pathway and the recruitment of inflammatory cells result in T lymphocyte migration, indicating electropermeabilization *per se* is able to recruit and trigger cells involved in antigen presentation (Chiarella et al. 2008b). Due to these immunostimulating effects, EP is now recognised as a good adjuvant (Chiarella et al. 2007), helpful in DNA vaccination for increasing the potency and safety of this therapeutic approach due to its property to induce a higher DNA uptake and its ability to stimulate both humoural and cellular immunity. At present, numerous findings are clarifying EP mechanism (Golzio et al. 2010), also showing the easy applicability of EP to large animals. In this view, many studies are concentrated to find the most appropriate and tolerable parameters that will make EP suitable for humans (Tjelle et al. 2008; Tjelle et al. 2006). To this purpose, various electroporating devices have been developed for animal and

Because these initial results seem promising, several clinical trials based on DNA vaccination assisted by electropermeabilization are under investigation and the efficacy and tolerability of EP will be deeply studied in the near future both in preclinical and clinical

The use of EP technology for DNA vaccination in the clinical settings takes advantage from the development of new protocols, which are effective in administrating the vaccine with

The type of EP device, intensity of electrical stimulations, number of electrical pulses and administrations, and choice of the target organ, are important parameters taken into

Because the need to develop non-invasive or minimally invasive genetic vaccination methods has become an important issue, several studies have been focussed on the needle-free injection of DNA vaccines. A recent patent, issued in 2007, reports the combination of needle-free injection and electroporation, demonstrating that this noninvasive strategy is sufficient to introduce the DNA vaccine in a form suitable for electrotransfer into a region of the host tissue. This needle-free injection may be used in combination with suitable non invasive electrode configurations (Hofmann 2007). Safe EP protocols for human disease therapies are under investigation and many preclinical studies are described in numerous works investigating the potential of EP in both

**3.2 Application of electrogene transfer in DNA vaccination protocols** 

the minimum discomfort and maximum tolerability for the patient.

consideration for the design of clinical protocols.

infectious and tumour diseases.

al. 2009).

human use (**Fig. 2**).

gene therapy protocols.

process is commonly known as electropermeabilization or electroporation (EP) (Chiarella et al 2010; Favard et al. 2007; Mir et al. 1999). The simultaneous publications in 1998 by Aihara and Miyazaki and Harrison and co-workers, demonstrated EP as being a more efficient method for gene transfer into muscle than the simple i.m. injection of DNA (Aihara and Miyazaki 1998; Harrison et al. 1998). The strategy is not only promising for enhancing the gene delivery of therapeutic proteins and drugs. Infectious disease, cancer gene therapy and chemotherapy are other fields of application, making electrochemogenetherapy relevant in a variety of research branches and promising in the gene therapy field (Mir 2008; Wells 2004). The advantage of DNA electrotransfer is dual. On the one hand, a high number of muscle cells are transfected with the DNA vaccine; on the other hand the damaged muscle cells release danger signals that favour antigen presenting cell recruitment, thus enhancing the immune response (Chiarella et al. 2008b). For this reason we consider the electropermeabilization such an important and very promising tool in the future of DNA vaccination therapy that it deserves a dedicated section of this book chapter.
