J Vet Sci.  2008 Dec;9(4):367-373. 10.4142/jvs.2008.9.4.367.

In vitro and in vivo gene therapy with CMV vector-mediated presumed dog beta-nerve growth factor in pyridoxine-induced neuropathy dogs

• Chung JY
• Choi JH
• Shin IS
• Choi EW
• Hwang CY
• Lee SK
• Youn HY

• KMID: 1104911
• DOI: http://doi.org/10.4142/jvs.2008.9.4.367

Abstract

Due to the therapeutic potential of gene therapy for neuronal injury, many studies of neurotrophic factors, vectors, and animal models have been performed. The presumed dog beta-nerve growth factor (pdbeta-NGF) was generated and cloned and its expression was confirmed in CHO cells. The recombinant pdbeta-NGF protein reacted with a human beta-NGF antibody and showed bioactivity in PC12 cells. The pdbeta-NGF was shown to have similar bioactivity to the dog beta-NGF. The recombinant pdbeta-NGF plasmid was administrated into the intrathecal space in the gene therapy group. Twenty-four hours after the vector inoculation, the gene therapy group and the positive control group were intoxicated with excess pyridoxine for seven days. Each morning throughout the test period, the dogs' body weight was taken and postural reaction assessments were made. Electrophysiological recordings were performed twice, once before the experiment and once after the test period. After the experimental period, histological analysis was performed. Dogs in the gene therapy group had no weight change and were normal in postural reaction assessments. Electrophysiological recordings were also normal for the gene therapy group. Histological analysis showed that neither the axons nor the myelin of the dorsal funiculus of L(4) were severely damaged in the gene therapy group. In addition, the dorsal root ganglia of L(4) and the peripheral nerves (sciatic nerve) did not experience severe degenerative changes in the gene therapy group. This study is the first to show the protective effect of NGF gene therapy in a dog model.

Keyword

dog; gene therapy; in vitro; in vivo; nerve growth factor; neuropathy

MeSH Terms

Amino Acid Sequence
Animals
Base Sequence
CHO Cells
Central Nervous System Diseases/chemically induced/therapy/*veterinary
Cloning, Molecular
Cricetinae
Cricetulus
Cytomegalovirus
Dog Diseases/*chemically induced/therapy
Dogs
Female
Gene Therapy/*veterinary
Genetic Vectors
Male
Molecular Sequence Data
Nerve Growth Factor/genetics/*metabolism/*therapeutic use
Pyridoxine/*toxicity

Figure

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  Fig. 1 The nucleotide and deduced amino acid sequence of the presumed dog β-NGF (pdβ-NGF) open reading frame (ORF) along with the primers used for cloning (underlined or boxes).

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  Fig. 2 PC12 cells were cultured with and without the filtered supernatant and photographed at ×100 magnification. (A) Negative control group. (B) Experimental group with cells showing neurite growth.

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  Fig. 3 (A) Normal dorsal funiculus of L4 in the negative control group. (B) Dorsal funiculus of L4, showing disruption of axons and myelin with vacuolation in the positive control group. (C) Dorsal funiculus of L4 showed occasionally swollen axons in the gene therapy group. H&E stain, ×200.

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  Fig. 4 (A) Normal dorsal root ganglia (DRG) of L4 in the negative control group. (B) DRG of L4 showed severe chromatolysis, vaculoation (arrowhead) and occasionally pyknotic nuclei and eosinophilic cytoplasm (arrows) in neurons in the positive control group. (C) DRG of L4 showed pyknotic nuclei and eosinophilic cytoplasm (arrows) in a few neurons in the gene therapy group. H&E stain, ×200.

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  Fig. 5 (A) Normal sciatic nerve of the negative control group. (B) Sciatic nerve having severe vacuolation (arrow) of the myelin in the positive control group. (C) Mild vacuolation (arrow) of the myelin in sciatic nerve of the gene therapy group. H&E stain, ×400.

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