Efficacy of nonviral gene transfer in the canine brain.
OBJECTIVE
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.
Oh
Seunguk
S
Department of Neurosurgery, University of Minnesota, St. Paul, USA.
Pluhar
G Elizabeth
GE
McNeil
Elizabeth A
EA
Kroeger
Kurt M
KM
Liu
Chunyan
C
Castro
Maria G
MG
Lowenstein
Pedro R
PR
Freese
Andrew
A
Ohlfest
John R
JR
D006516:Adenoviruses, Canine / Q000235:genetics*; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000821:virology; D001774:Blood Chemical Analysis; D001921:Brain / Q000166:cytology / Q000201:enzymology / Q000821:virology; D001932:Brain Neoplasms / Q000628:therapy; D020805:Central Nervous System Viral Diseases / Q000235:genetics / Q000473:pathology / Q000821:virology; D004285:Dogs; D005240:Feasibility Studies; D018014:Gene Transfer Techniques / Q000295:instrumentation*; D015316:Genetic Therapy / Q000295:instrumentation*; D005822:Genetic Vectors / Q000235:genetics; D005910:Glioma / Q000628:therapy; D007249:Inflammation / Q000473:pathology / Q000821:virology; D007963:Leukocytes, Mononuclear / Q000378:metabolism / Q000821:virology; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000166:cytology / Q000378:metabolism / Q000821:virology; D010957:Plasmids / Q000235:genetics* / Q000502:physiology; D011094:Polyethyleneimine / Q000627:therapeutic use; D014161:Transduction, Genetic / Q000379:methods; D019444:Vaccines, DNA / Q000235:genetics
D019444:Vaccines, DNA; D011094:Polyethyleneimine; D049409:Luciferases, Firefly