**1. Introduction**

596 Non-Viral Gene Therapy

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The introduction of genes into the organism or the regulation of the expression of endogenous genes has emerged in the last decade as a very potent strategy for correcting monogenic inherited diseases, treating acute disorders, and slowing down the progression of diseases without known cure. In addition it constitutes an important tool for research, which has been widely used and has contributed to show the mechanisms behind several physiological processes and pathologies.

Adequate carriers able to transfer DNA or RNA into target cells have been largely explored. However, this is an area under continuous expansion as there is no ideal vector suitable for all applications. In fact, no individual vector will meet all the characteristics for a perfect or ideal vector, as many of the needs are different and even contradictory. For example, immunogenicity is in most cases an undesirable side effect, while it is a valuable property when treating tumours as it contributes to their clearance. Another example of contradictory needs of one single vector would be the capacity of a vector to determine the overexpression of the transgenic protein for life. This would be an essential property for the treatment of inherited diseases produced by the lack of a particular protein, however for the treatment of acute injuries the lifelong expression of a therapeutic protein will probably be deleterious. Moreover, some vectors do not transduce post-mitotic cells like neurons or muscle fibres, which is a drawback for targeting these cell types but may be an advantage for the targeting of cancer cells. Thus, there is a need for diverse type of vectors for diverse therapeutic or experimental paradigms, and in particular versatile tuneable vectors would be very interesting. Moreover, several basic problems with the known vectors persist, like toxicity, oncogenicity, immunogenicity, low transfection efficiency, or poor bioavailability, which need further consideration and efforts.

Due to their natural efficiency, viruses have been modified to act as vectors, and they have shown a good degree of success. Non-viral vectors have also been developed by combining several properties necessary for transfection: nucleic acid attachment and condensation, cell attachment, cell entry, endosomal escape, intracellular trafficking, nuclear entry, and nucleic acid release. Some of these vectors are quite simple, as the ones formed by the combination of nucleic acids and lipid components or other carriers like polyethylene glycol (PEG). Others include the previous components but have in addition attached targeting molecules like antibodies, enabling these vectors to preferentially transfect a given tissue. In fact even

Modular Multifunctional Protein Vectors for Gene Therapy 599

Surfactant Protein A-poly-lysine modular vector or a transferrin-poly-lysine modular vector (Ross *et al.* 1998). Naturally DNA condensing proteins have also been used for the construction of modular vectors. For instance, Histones condense plasmid DNA and protects it from endonucleases, being the lysine-rich H1 Histone the most effective one (Pyhtila *et al.* 1976). Moreover, some nuclear localization signals like the NLS peptide from SV40 virus large T-antigen are lysine-rich peptides that when used as a tetramer can efficiently condense DNA without loosing its nuclear localization properties (Ritter *et* 

When a viral or non-viral gene therapy vector is injected intravenously, most of the vectors will localize mainly in the liver but also in the kidneys, lungs and spleen. While this is normally a problem to circumvent for most gene therapy applications, it constitutes an advantage for the expression of molecules in the liver. There are many fetal metabolic diseases resulting from a defect or a deciency of hepatocyte-derived proteins. Moreover, the liver can be considered as a platform to produce various proteins secreted into the blood. Therefore, many pioneer studies focused on the development of more efficient gene delivery systems for the introduction of therapeutic genes selectively into hepatocytes (Wu and Wu 1988). Intravenously injected plasmids are cleared from the circulation by the liver non-parenchymal cells by a scavenging receptor mediated mechanism (Kawabata *et al.* 1995). When Nishikawa and colleagues administered naked 32PDNA into the tail vein of mice, about 40% and 10% of the radioactivity rapidly accumulated in the liver and kidneys, respectively (Nishikawa *et al.* 2000b). Again, the main cell-types targeted were the liver nonparenchymal cells: Kupffer cells and endothelial cells. When they injected a vector composed of 32PDNA/polyornithine, little effect on the distribution of the DNA was observed. However, the injection of the 32PDNA/Gal-pOrn galactose-mediated hepatocytetargeting vector induced a 60% hepatic accumulation of radioactivity, but more interestingly, most of the targeted cells were now hepatocytes instead of Kupffer or endothelial cells. The same effect was observed at the level of luciferase transgene expression, indicating that the DNA/Gal-pOrn vector was not only able to adhere and enter

Many different domains of known proteins and sugars have been used for cell targeting of modular vectors, like galactose (Wu and Wu 1987), transferrin (Wagner *et al.* 1990), foot-andmouth disease virus integrin interacting peptide (Aris *et al.* 2000; Aris and Villaverde 2003; Domingo-Espín *et al.* 2011), nerve growth factor (Ma *et al.* 2004; Zeng *et al.* 2004), surfactant protein A (Ross *et al.* 1995), rabies virus glycoprotein (Kumar *et al.* 2007), tetanus toxin fragment Hc (Box *et al.* 2003; Knight *et al.* 1999), cholera toxin b chain (Barrett *et al.* 2004), and neurotensin (Navarro-Quiroga *et al.* 2002). In an interesting study, Arango-Rodríguez and colleagues showed that they could target only substantia nigra neurotensin high affinity receptor positive neurons by means of a modular vector that displayed neurotensin, while no other neurons were transfected (Arango-Rodriguez *et al.* 2006). *In vivo*, many of these targeting systems have shown success (see Table 1). An additional interesting targeting strategy is the use of antibodies (Berhanu and Rush 2008; Buschle *et al.* 1995; Thurnher *et al.* 1994). For instance, the use of the 1E3 antibody against the Tn antigen expressed on many carcinomas coupled to polylysine induced an important increase in the transfection of a cancer cell line (Thurnher *et al.* 1994). Another vector, named fkAbp75-ipr, possess several

*al.* 2003).

**2.2 Cell attachment and cell targeting** 

preferentially into hepatocytes, but it could also transfect them.

magnetic fields have been used to concentrate suitable engineered vectors to a given area (Corchero and Villaverde 2009).

An interesting type of non-viral vectors is the one based on multifunctional proteins (Aris and Villaverde 2004; Mastrobattista *et al.* 2006). The combination of functional domains in a single polypeptide is a simple yet powerful approach for the development of vectors suitable for gene therapy. In fact, this approach has generated the first prototypes of modular protein gene therapy vectors. Three general methods have been used for the engineering of these molecules: i) production of a recombinant protein by the direct fusion of the functional domains; ii) production of a recombinant protein by combining a known scaffold protein and several functional domains inserted into exposed regions of the scaffold protein; and iii) chemical conjugation of functional domains and proteins. Many of these vectors can be produced recombinantly, generating reproducible and stable stocks appropriate for the formulation of clinically usable drugs. Moreover, the modular nature of these versatile vectors enables the combination of different domains to fulfil the changing requirements of pathological end experimental situations.
