Biologic Response Human Intervertebral Disc Cells - Gene Therapy Cocktail, Spine 2008

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Pubmed: 18622355

SPINE Volume 33, Number 17, pp 1850–1855
© 2008, Lippincott Williams & Wilkins

Biologic Response of Human Intervertebral Disc Cells to Gene Therapy Cocktail

Seong-Hwan Moon, MD,* Kotaro Nishida, MD,† Lars G. Gilbertson, PhD,†
Hwan-Mo Lee, MD,* Hyang Kim, MSc,* Ronald A. Hall, MD,† Paul D. Robbins, PhD,‡
and James D. Kang, MD†

*Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea;
†Department of Orthopaedic Surgery, Ferguson Laboratory for Orthopaedic Research, University of Pittsburgh Medical Center, Pittsburgh, PA; and
‡Department of Molecular Genetics and Biochemistry, university of Pittsburgh, Pittsburgh, PA.

Study Design
In vitro experiment using human intervertebral disc (IVD) cells and adenovirus-therapeutic gene constructs.

Objective
To examine the biologic effect of “cocktail” therapeutic gene transfer to human IVD cells in three dimensional cultures.

Summary of Background Data
Gene therapy is regarded as a potential option for the treatment of degenerative disc disease. Although various anabolic genes have previously been introduced for this purpose, cocktail gene transfer of anabolic genes to IVD cells has never been attempted.

Methods
Human IVDs were harvested during surgical disc procedures and cultured. We prepared recombinant adenovirus constructs bearing the TGF-1 gene (Ad/TGF-b1), the IGF-1 gene (Ad/IGF-1), and the BMP-2 gene (Ad/BMP-2). Transgene expression was detected by luciferase assays, enzyme linked immunosorbent assays, and Western blot analysis. Newly synthesized proteoglycan was measured by 35S-sulfate incorporation on Sephadex G-25M in PD 10 columns. Human IVD cells were transduced by single, double, and triple combination of Ad/TGF-B1, Ad/IGF-1, Ad/BMP-2 with an MOI of 75, then cultured threedimensionally in alginate beads.

Results
Transgene expression was detected at 18 hours after viral transduction. IVD cultures with Ad/TGF-B1, Ad/IGF-1, Ad/BMP-2 (MOI of 75) showed 2.9, 1.8, and 1.9 fold increases, respectively, in proteoglycan synthesis compared to control. Human IVD cultures with double gene combination (MOI of 75) showed 3.2 to 3.9 fold increases of proteoglycan synthesis. Lastly, Human IVD cultures with triple gene combination (TGF-1IGF-1BMP-2 genes with an MOI of 75) transfer demonstrated 4.7 fold increase in proteoglycan synthesis compared control.

Conclusion
Combination or “cocktail” gene therapy offers a promising mechanism for maximizing matrix synthesis with low dose of adenoviral mixtures, circumventing systemic, local toxic effect, and immune response.


Key Points
● Therapeutic gene transfer of the TGF-
1, IGF-1, and BMP-2 genes upregulated proteoglycan synthesis in human intervertebral disc cells

● Combination “cocktail” gene transfer (double genes, triple genes) to human intervertebral disc cells demonstrated enhanced proteoglycan synthesis compared to single therapeutic gene transfer.

● Combination “cocktail” gene therapy renders a mechanism of enhanced matrix synthesis, while circumventing virus-induced toxicity and immune response.

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