Efficient and stable gene expression in rabbit intervertebral disc cells transduced with a recombinant baculovirus vector.
METHODS
A recombinant baculovirus vector expressing GFP (Ac-CMV-GFP) was tested in rabbit intervertebral disc cells cultured in monolayer in vitro. Direct infection of intervertebral disc cells was then assessed in vivo.


OBJECTIVE
To test the efficacy of the baculovirus-mediated marked gene transfer to rabbit intervertebral disc cells, and the expression of the transgene in vitro and in vivo.


BACKGROUND
The potential application of gene therapy for the treatment of degenerative disc disease has been an area of exciting and active research. Many efforts have been made toward the construction of delivery vectors using mammalian viruses, such as adenovirus, adeno-associated virus, retrovirus, and herpesvirus. However, recently, baculovirus-derived vectors have emerged as possible tools for gene transfer into mammalian cells in vitro and in vivo but, to our knowledge, have never been tested in an intervertebral disc cell.


METHODS
The intervertebral disc cells cultured in monolayer were treated with 6 different doses of baculovirus carrying the green fluorescence protein gene (Ac-CMV-GFP). Fluorescence microscopy and flow cytometry were used to analyze transgene expression. The Autographa californica nucleopolyhedrovirus/GFP virus was then injected directly into the intervertebral discs of 8 rabbits at 7, 13, 20, and 28 days after injection. The nucleus pulposus tissues of injected discs were evaluated immediately by fluorescence microscopy for GFP expression.


RESULTS
A dose of Ac-CMV-GFP at a multiplicity of infection of 200 achieved the highest transduction ratio (approximately 87% of nucleus pulposus cells) and long-term expression without any toxicity to the cells. In vivo assay showed that Ac-CMV-GFP could also mediate GFP expression in rabbit intervertebral disc cells without inducing any symptoms. The GFP expression level at 7 days after transduction was significantly higher than at 21 and 28 days after treatment.


CONCLUSIONS
Baculovirus can transfer exogenous genes into rabbit nucleus pulposus cells at a high efficiency, and safely both in vitro and in vivo. The results suggest that baculoviruses might be useful tools as gene therapy vectors for intervertebral disc diseases.
Liu
Xiaoyun
X
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, People's Republic of China.
Li
Kanghua
K
Song
Jianhua
J
Liang
Changyong
C
Wang
Xiyang
X
Chen
Xinwen
X
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics*; D007403:Intervertebral Disc / Q000166:cytology / Q000378:metabolism*; D011817:Rabbits; D014161:Transduction, Genetic / Q000379:methods*