**3.1 Naked plasmid transfection**

152 Non-Viral Gene Therapy

Fig. 1. Rationale for Gene Therapy in Lysosomal Storage Disorders: Cross-correction. The upper part of the figure shows two cells from a patient with LSD, with large lysosomes (green) due to accumulation of undegraded material. The gene transfer of a normal copy of the missing enzyme (red structure in the nucleus), allows the enzyme (red triangles) to be produced, and degrade the material accumulated in the lysosomes. Part of the enzyme is secreted from the recombinant cell and is captured by neighbouring cells via the mannose-6 phosphate receptors (light blue structures located on the cell membrane), reaching the lysosomes and being able to correct their phenotype, in a process called cross-correction. Note that the cell on the right was never transduced, but is able to capture the enzyme from

the circulation or from the neighbouring cell.

The direct injection of a plasmid containing the gene of interest and the regulatory mechanisms to ensure its expression is the simplest form of gene therapy. It has some advantages over the virus mediated gene transfer systems. First, DNA preparation is simple and can be performed at relatively low cost. In addition, the safety concerns are much lower and large amounts of DNA can be transferred. However, the major limitation of this method is that it requires a local administration and the level of transgene expression is relatively low and restricted to the injection site (Glover et al., 2005).

Hydrodynamic injection (Liu et al, 1999) is an experimental method capable to achieve efficient gene transfer and high level of transgene expression by systemic administration. In this procedure, a large volume of saline containing plasmid DNA is injected in a short period of time. The large volume and high injection rate forces the DNA solution into the liver, probably due to the permeability of liver fenestrae. A small hepatotoxicity, probably due to the large volume of saline, is observed and resolves within a few days. Even though this procedure is widely used in mice by tail vein injection, its feasibility has been demonstrated in larger animal models using a balloon occlusion catheter-based method to mimic hydrodynamic injection (Brunetti-Pierri et al, 2007; Kamimura et al, 2009).

This approach has been used in a proof-of-concept study to show the importance of coexpression of the formylglycine-generating enzyme for synthesis and secretion of functional Arylsulfatase A in a mouse model of Metachromatic Leukodystrophy (Takakusaki et al., 2005). This enzyme is a posttranslational modifying enzyme essential for activating multiple forms of sulfatases including Arylsulfatase A and therefore limits the amount of functional enzyme that can be secreted from transduced cells.

It has also been used in Mucopolysaccharidosis (MPS) type I (Camassola et al, 2005) and type VII (Richard et al, 2009). In the MPS I animals, storage content was reduced and enzyme activity was elevated in the liver and spleen. For the MPS VII model, a beneficial effect on the pathology was also observed, as liver-directed gene transfer led to the correction of secondary enzymatic elevations and to the reduction of GAGs storage in peripheral tissues and brain, as well as to histological correction in many tissues.
