Therapeutic effects of adenovirus-mediated growth and differentiation factor-5 in a mice disc degeneration model induced by annulus needle puncture.
BACKGROUND
The therapeutic strategies that have thus far been used for the treatment of intervertebral disc degeneration (IDD) have focused on relieving the symptoms, although reversal of the degeneration remains an important challenge for the effective treatment of IDD. Growth and differentiation factor-5 (GDF5), of which deficiency leads to early disc degeneration changes, has the potential to increase proliferation of disc cells and expression of extracellular matrix proteins.


OBJECTIVE
The purpose of the study was to develop a lumbar disc degeneration model in mice and determine the effect of adenoviral GDF5 gene therapy.


METHODS
The study design was to compare the degeneration changes of discs punctured by different-size needles to develop a mice lumbar disc degeneration model and to evaluate the effects of in vivo gene therapy for the mice disc degeneration model by an adenoviral vector carrying GDF5 gene.


METHODS
A lumbar disc degeneration model was developed by needle punctures to the discs in Balb/c mice. Afterward, a gene therapy treatment to disc degeneration was evaluated. Two of the mice lumbar discs were randomly chosen to be punctured by a 30-gauge needle and then injected with adenovirus that had been engineered to express either the luciferase gene (Ad-Luc) or the GDF5 gene (Ad-GDF5). Animals were analyzed by bioluminescent imaging, radiographic, and magnetic resonance imaging (MRI) scanning, then sacrificed at 1, 2, 4, or 8 weeks after operation, and subjected to histological and biochemical assays.


RESULTS
By the detection of T2-weighted MRI scanning and histological study, the degeneration was found in all of the discs punctured by different-size needles. But the development of the degeneration in the discs injured by the 30-gauge needle was more reliable and moderate compared with that in other groups. The detection of luciferase activity by bioluminescent imaging revealed that adenovirus survived and the introduced genes were expressed over 6 weeks after injection. There were no T2-weighted MRI signals in the mice injected with either Ad-Luc or Ad-GDF5 up to 4 weeks after operation. At 6 and 8 weeks, T2-weighted signals were detected in the Ad-GDF5 group but none in the Ad-Luc control group. The percent disc height index (%DHI) was significantly decreased (approximately 20%) by 1 week after injury in both groups, indicating the development of disc degeneration. At 2 weeks, the %DHI in the mice injected with Ad-GDF5 increased significantly compared with that of the mice injected with Ad-Luc; the increase was sustained for the rest of the experiment period. The disc histology treated with Ad-GDF5 was improved compared with that in the control group. Glycosaminoglycan (GAG) levels were significantly decreased in the Ad-Luc injection group since 2 weeks after injury, and the DNA content had diminished by 4 weeks after the operation. In contrast, in the discs injected with Ad-GDF5, there was no decrease in the GAG and DNA levels after injury throughout the 8-week treatment period.


CONCLUSIONS
Disc degeneration animal model can be developed by using needle puncture to the discs in mice. The adenovirus is an effective vehicle for gene delivery with rapid and prolonged expression of target protein and resulting improvement in markers of disc degeneration. Ad-GDF5 gene therapy could restore the functions of injured discs and has the potential to be an effective treatment.
Liang
Haixiang
H
Department of Orthopaedic Surgery, School of Medicine, University of Virginia, Charlottesville, VA 22908, USA. xl2n@virginia.edu
Ma
Shen-Ying
SY
Feng
Gang
G
Shen
Francis H
FH
Joshua Li
Xudong
X
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002455:Cell Division / Q000502:physiology; D004195:Disease Models, Animal; D016326:Extracellular Matrix Proteins / Q000502:physiology; D015316:Genetic Therapy / Q000379:methods*; D055428:Growth Differentiation Factor 5 / Q000235:genetics*; D007403:Intervertebral Disc / Q000473:pathology / Q000502:physiology; D055959:Intervertebral Disc Degeneration / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D008279:Magnetic Resonance Imaging; D051379:Mice; D008807:Mice, Inbred BALB C; D016602:Needlestick Injuries
D016326:Extracellular Matrix Proteins; C525894:Gdf5 protein, mouse; D055428:Growth Differentiation Factor 5