Iso 360 360

num be r/plasm id

+ 1 inM "luialliiont;

+ phage 74 DNA

Figure 5 Agarose gel electrophoresis shows complex formation between DNA, cationic detergent, and compound PEG-fol. Plasmid DNA (30 mM bp, lane 1) was mixed with increasing amounts of PEG-fol (1 mM, lane 2 and 2 mM lanes 3, 4); lane 4: DNA-PEG complexes were incubated for 15 min with 1 mM glutation. Lanes 5-8: nanometric (C14CO)2/DNA particles were prepared as in Fig. 4. After overnight incubation, compound PEG-fol (lanes 6-8) was added to the particles; lane 8: 1 h incubation of the complexes with phage T4 DNA. Image: Transmission electron microscopy picture of the final (C14CO)2-DNA-PEG-fol complexes shows monomolecu-lar DNA condensation into compact particles. See the color insert for a color version of this figure.

toxic, and versatile agents for in vivo gene delivery by a number of routes. These commercially available polymers have the added advantage that their relatively high in vivo performance is unhindered by immune effects. Like other synthetic vectors, PEI provides the extra benefit of facility of use for complexing and condensing DNA. This is turn leads to ease of testing DNA constructs and scaling up of protocols. Preclinical data toward gene therapy approaches of AIDS (48), head and neck cancer (49,50), and pancreas (51,52) and ovarian cancer (53) disseminations in the peritoneal cavity are encouraging.

That PEI provides relatively high levels of transfection in a number of target organs, by various delivery routes, prompts a series of questions. Given that the intracellular barriers to delivery are probably similar for in vivo, ex vivo, and in vitro delivery, the main point of inquiry is how DNA-PEI complexes overcome the series of anatomical and physiological barriers that hinder delivery of functional DNA to target cells in vivo. A key point would appear to be the small size of particles obtained when DNA is condensed in a defined me dium. PEI-DNA complexes can be formulated that have a size range close to that of viruses (50 nm, Fig. 7) and with a charge ratio that does not hamper biodistribution and yet maintains efficient transfection. According to the choice of formulation solution, small particles can be obtained whether branched (25 kDa) or linear (22 kDa) are chosen. Using linear 22 kDa PEI to complex DNA in salt solutions gives quite large particles (> 1 ^m) that work exceedingly well in vitro (47). However, formulation with the same 22 kDa in an equi-osmotic but salt-free solution of 5% glucose produces particles with mean size ranging between 30 and 60 nm (54) (Fig. 7). Particles of <100 nm with the branched 25-kDa form can be obtained in 150 mM NaCl (23,55). However, despite the roughly similar sizes of particles formulated in appropriate conditions (< 100 nm), there are quite clear-cut differences in the performances obtained in animal models (47,56) with linear 22-KDa PEI consistently providing the greatest efficiency. Improved transfection of resting cells with linear PEI (46) may be the clue to this observation. The more recent findings on the better performance of the complexes formulated in

Figure 6 (C14CO)2-DNA-PEG-fol complexes specifically bind to folate receptor-expressing KB cells. Cells were incubated for 3 h with fluorescent DNA complexes and sorted by cytometry according to their fluorescence. Only cells overexpressing the folic acid receptor (right graph) show an increased fluorescence (filled trace) over nontreated cells (hollow trace).

Figure 6 (C14CO)2-DNA-PEG-fol complexes specifically bind to folate receptor-expressing KB cells. Cells were incubated for 3 h with fluorescent DNA complexes and sorted by cytometry according to their fluorescence. Only cells overexpressing the folic acid receptor (right graph) show an increased fluorescence (filled trace) over nontreated cells (hollow trace).

salt-free conditions corroborate early work injecting DNA/ PEI particles into the brains of adult mice (57).

The description of the small size of particles formulated in salt-free conditions with 22-KDa PEI was accompanied by the demonstration that these particles were stable in cerebro-spinal fluid and led to a large diffusion of active complexes throughout the brain following intraventricular injection (54). This in turn led to the application of the methodology in a number of different settings, including the central nervous system, the respiratory tract, or tumors.

A. Central Nervous System

PEI has been used to deliver DNA into the CNS of rodents both for proof of principle and to analyze physiological functions [for instance, see (58,59)], including regulation of gene expression (promoter studies) and function of proteins (mainly transcription factors and transporter proteins). As concerns the basic optimization of the methodology, keeping concentration of DNA <1 ^g/^L increases yield (60). Indeed, in the newborn mouse brain, one can obtain up to 2.107 RLU/mg protein when injecting a mere 20 ng DNA. Such conditions also allow for diffusion of particles not only within the intra-ventricular space, but also through the ependymal cell layer lining the ventricles and through 4 or 5 cells layers beyond it (54). Most recently, using the same intraventricular delivery route, it has been shown that gene delivery with unmodified

PEI permits privileged targeting of the neuronal stem cell population in adult mice (61) (Fig. 8).

Similarly, using low DNA concentrations (0.25 ^g/^L) and a low charge ratio (3-6 N/P) allowed Marthres and collaborators (62) to obtain increased expression of the dopamine transporter throughout the entire substance nigra (a brain nucleus with a diameter of about 5 mm) in adult rats. Significant increases in binding and uptake were found in all the brain areas receiving neuronal projections from the brain structure transfected (sustantia nigra). This finding is important because it underlines the fact that postmitotic neurons can be transfected by this method, in agreement with the related properties of linear PEI (46-63,64). Equivalent results in terms of efficiency of expression in neurons of the adult rat were obtained when injecting plasmids encoding the serotonin transporter into the raphe nucleus of adult rats (65). In this study, effects of modifiying gene expression were also assessed on behavior (sleep/wake cycle), and significant and appropriate changes found for weeks following transfection. Yet another means of gene delivery to the brain with PEI was demonstrated by Wang et al. (66). These authors observed that following injection of PEI-DNA complexes into the tongue muscle of mice, significant reporter gene activity could be found in the brain stem. This finding suggests that PEI-DNA complexes can cross the neuromuscular junction and enter the brain by retrograde axonal transport, thus bypassing the blood-brain

Figure 7 Electron micrographs of PEI 22-kDa-DNA complexes prepared in Nacl 0.15 M (A, N/P = 2) or in 5% (w/w) glucose (B, N/P = 5); bar represent 100 nm.

barrier in a manner similar to vectors based on the proteolytic C fragment of tetanus toxin (TTC peptide) (67).

B. Delivery to Respiratory Tract

Gene therapy to the lung could be exploited for both genetic and acquired diseases. However, like the brain, any therapeutic approach for the respiratory tract must take into account the heterogeneity of the cellular targets in the lung: epithelial cells, alveolar cells, vascular cells, and serous cells in the submucosal glands, etc. Two main routes can be used to introduce genes into pulmonary tissue: via the airways or through the circulation by systemic injection.

Both branched and linear PEIs have been tested with moderate success for delivering DNA by the tracheal route. Branched 25-kDa PEI gave higher levels of transgene expression than fractured polyamidoamine dendrimers especially when DNA-PEI complexes were prepared in water (68). Another group has tried nebulization of PEI-DNA complexes prepared in water (69). Their data show that PEI can protect DNA during nebulization and therefore give enhanced transgene expression. In a further analysis, they show that nebulization with 5% CO2 improves transfection over that seen with air (70). Yet, in a recent study (71), PEI was shown to give exceptionally high (>1 ng luciferase/mg protein, ca. 1%-5% transfected tracheal cells) and more durable expression (>4 days) following slow instillation of the complexes into the mouse lung.

Following the demonstration that in 5% glucose DNA com-plexed with 22-KDaPEIprovides small particles showing good stability and diffusibility in the brain, it was logical to test whether these particles would be stable enough to provide transfection of various organs following systemic injection. To this end, we analyzed transfection obtained in different tissues of the mouse after injection of varying quantities of DNA com-plexed with different N/P ratio through the tail vein. It was found that in all cases the lung showed the highest levels of transgene expression with both luciferase and the p-galactosi-dase genes (72,73). Anatomical analysis revealed pulmonary cells to be transfected and no signs of lesions or toxic effects. This suggested that the particles were crossing the pulmonary capillary barrier, although double labeling was not carried out. Later work showing the transfection of pulmonary cells was confirmed by double immunostaining with appropriate markers and passage of the pulmonary epithelium was shown to be both rapid and nontraumatic (74). Bragonzi et al. (56) used this model with GFP expressing vectors and tagged complexes to further the analysis. They also compared transfection performances of polyplexes formed with branched and linear PEIs to that of two lipoplexes. The most efficient vector for delivery by this route was linear 22 kDa. DOTAP gave reasonable levels of expression with naked DNA, GL-67A, and 25kDa PEI showing negligible transfection.

C. Tumor Targeting

Obviously, another major field of application for gene transfer technology is the treatment of tumors. Unmodified or modified PEI has been used with the aim of targeting lung metasta-

Figure 8 Privileged transfection of neuronal stem cells in adult mice with PEI. (A) In the adult CNS, intraventricular injection of PEI-DNA complexes results in transgene expression (P-gal positive blue cells), mainly on the striatal aspect of the lateral ventricles. (B-D) Identification of the transfected cells revealed a preferential transfection of the adult neural stem cells of the subventricular zone and their immediate progeny. (B) Positive cell with the typical morphology of astrocyte. (C) Confocal microscopy confirm GFP reporter gene expression in GFAP (marker of astrocytes)-positive cells in the subependymal layer. (D) Transfected cells (lacZ-positive cell) in this area also express Nestin, a marker of neural progenitors. Bar: (A) 200 ^m; (B-D) 12 ^m. St., striatum; LV, lateral ventricle. See the color insert for a color version of this figure.

Figure 8 Privileged transfection of neuronal stem cells in adult mice with PEI. (A) In the adult CNS, intraventricular injection of PEI-DNA complexes results in transgene expression (P-gal positive blue cells), mainly on the striatal aspect of the lateral ventricles. (B-D) Identification of the transfected cells revealed a preferential transfection of the adult neural stem cells of the subventricular zone and their immediate progeny. (B) Positive cell with the typical morphology of astrocyte. (C) Confocal microscopy confirm GFP reporter gene expression in GFAP (marker of astrocytes)-positive cells in the subependymal layer. (D) Transfected cells (lacZ-positive cell) in this area also express Nestin, a marker of neural progenitors. Bar: (A) 200 ^m; (B-D) 12 ^m. St., striatum; LV, lateral ventricle. See the color insert for a color version of this figure.

ses or subcutaneous tumors (75). Various modes of delivery have been tested to this end: direct intratumoral of unmodified 22-kDa PEI (76), intravenous injection of transferrin-conju-gated PEI (21) or even aerosolized PEI-DNA complexes to reach lung tumors (70). Interestingly, it is in this field of endeavor that multicomponent approach has yielded the best results. Kircheis et al. (43) shielded either 25-kDa or 22-kDa PEI-DNA complexes by covalently incorporating transferrin and used the complexes for systemic injection of mice bearing subcutaneous tumors. The presence of transferrin on the com plexes provided a preferential transfection of the tumors rather than the lung.

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