Efficacy of nonviral gene transfer in the canine brain

Abstract

Object: The purpose of this study was to evaluate the gene transfer capability and tolerability of plasmid DNA/polyethylenimine (PEI) complexes in comparison with adenovirus and naked plasmid DNA in the canine brain.

Methods: Plasmid or adenoviral vectors encoding firefly luciferase were injected directly into the cerebral parenchyma of five adult dogs at varying doses and volumes. Serial physical and neurological examinations, as well as blood and cerebrospinal fluid (CSF) analyses, were conducted before and after the surgery for 3 days. Three days after gene delivery, a luciferase activity assay and immunofluorescence analysis were used to test the brain tissue for gene expression.

Results: Injection into the brain parenchyma resulted in gene transfer throughout the cerebrum with every vector tested. Luciferase expression was highest when adenovirus vectors were used. Injection of plasmid DNA/PEI complexes and naked DNA resulted in similar levels of luciferase expression, which were on average 0.5 to 1.5% of the expression achieved with adenovirus vectors. Immunofluorescent microscopy analysis revealed that plasmid DNA/PEI complexes transduced mainly neurons, whereas adenovirus transduced mainly astrocytes. No significant acute side effects or neurological complications were observed in any of the dogs. Mononuclear cell counts significantly increased in the CSF after adenovirus injection and modestly increased after injection of plasmid DNA/PEI complexes, suggesting that a mild, acute inflammatory response occurred in the central nervous system (CNS).

Conclusions: Compared with rodent models that are limited by very small brains, the dog is an excellent preclinical model in which to assess the distribution and safety of emerging gene transfer technologies. In this study, short-term gene transfer was evaluated as a prelude to long-term expression and safety studies. The authors conclude that the viral and nonviral vectors tested were well tolerated and effective at mediating gene transfer throughout a large portion of the canine brain. The nonviral plasmid vectors were less effective than adenovirus, yet they still achieved appreciable gene expression levels. Due to reduced gene transfer efficiency relative to viral vectors, nonviral vectors may be most useful when the expressed protein is secreted or exerts a bystander effect. Nonviral vectors offer an alternative means to genetically modify cells within the CNS of large mammals.

FIG. 1

A: Image showing that all plasmid and adenoviral vectors were injected 2 cm beneath the surface of the cortex. B: Schematic showing the bolus injection sites (arrows). One dog was given five bilateral injections of RAdLuc (5 μl/site). Two dogs were given five bilateral injections of plasmid DNA, naked (one animal) or complexed in PEI (one animal) in a volume of 20 μl/site. C: Schematic demonstrating the vector infusion sites (arrows) for slow infusion. Two dogs were given one bilateral injection of DNA/PEI complexes (200 μl/site over 10 minutes). D: Schematic depicting the brain processing for luciferase assays. The left and right cerebral hemispheres were separated. Each hemisphere was sliced into three arbitrary sections (dorsal, medial, and ventral) of equal thickness (dotted lines). Each slice of tissue was then homogenized and assayed for luciferase expression. The cerebellum and brainstem were discarded before assay (solid line).

FIG. 2

Graphs. Upper: Dogs were bilaterally injected intrace-rebrally with 8.19 × 10⁸ pfu of RAdLuc, plasmid DNA/PEI complexes (50 μg; N/P ratio = 6), or naked DNA (50 μg). One vector-treated cerebral hemisphere obtained in each dog was sliced into three sections (dorsal, medial, and ventral) of equal size, homogenized, and assayed for luciferase activity. Lower: Note the luciferase activity of pooled lysates from the entire cerebral hemisphere (normalized to expression in adenovirally injected brain). RLU = relative light unit; s = second.

FIG. 3

Photomicrographs and bar graph. A: Dogs received a bilateral injection of 100 μg plasmid DNA/PEI complexes or 8.19 × 10⁸ pfu adenovirus encoding luciferase (Luc) into each cerebral hemisphere. Luciferase immunofluorescence staining was conducted along with markers for mature neurons (NeuN) and astrocytes (GFAP). Adenovirus transfected mainly astrocytes (left column), whereas plasmids transfected mainly neurons (right column). Original magnification × 40. B: Representative images of luciferase immunofluorescence. Staining in the adenovirus-treated dog was most intense in areas surrounding the needle track (left). Luciferase-positive cells were also noted several centimeters away from the injection sites lining the ventricles (right). Original magnification × 20. C: Luciferase-positive cells were counted in serial brain sections obtained in dogs treated with adenovirus or DNA/PEI complexes (four sections/dog brain). The distance from the needle track in any direction was noted and the majority of positive cells were within 1 mm of the needle track. There were significantly more luciferase-positive cells near the needle track in the adenovirus-treated dog than the DNA/PEI complex–treated dog. Error bars represent the standard deviations. *p ≤ 0.05 (Student t-test).

FIG. 4

Graph demonstrating rectal body temperatures measured before and every 12 hours after gene delivery.

FIG. 5

Photomicrographs showing H & E staining of needle track sites in adenovirally and DNA/PEI-injected canine brains. A high number of mononuclear cells were observed infiltrating needle track areas in the adenovirally injected canine brain (left column). The DNA/PEI complex–injected brain showed less infiltration of inflammatory cells (right column). Original magnification appears on the images.