Fatty Acid Mediated Transfection of Potentially Therapeutic Genes in Human Intervertebral Disc Chondrocytes

Funding:

North American Spine Society

Principal Investigator:

Paul A. Anderson, MD

Lab Website:

(Lab website not available at this time)

Project Summary:

Objectives:

1. To identify an optimal non-viral reagent and dose resulting in low toxicity and high efficency for transfection of human intervertebral thoracolumbar disc chondrocytes.
2. To evaluate human chondrocyte gene expression of SOX-9 and BMP-7 when transfected using a non-viral reagent compared to controls and adenoviral tranfection.

Summary of the Background Data:

Recent studies suggest that exogenous genes such as Sox9 and BMP-7 upregulate Type II collagen and proteoglycan synthesis. Interventions that augment type II collagen production by intervertebral thoracolumbar disc cells may represent an important new therapeutic modality for patients with degenerative disc disease. Although well established, these chondrogenic effects were conducted using an adenoviral vector. Adenoviral vectors pose significant immunologic and toxicity concerns. Lipid based reagents have been used to successfully transfect articular chondrocytes in vitro. Our initial pilot study documents successful transfection of human intervertebral thoracolumbar disc chondrocytes using a lipid based reagent.

Experimental Design:

Human disc tissues taken during routine discectomies will be treated and cultured. These disc cells with then be exposed to marker genes packaged in lipid based vectors. Cell cultures will be evaluated for evidence of marker gene expression. After identifying an optimal reagent, we will titrate ratios and quantities of DNA versus lipid reagent to minimize toxicity while maximizing transfection rate and gene expression. Using the optimal vector and conditions, we will transfect human chondrocytes with the SOX-9 and BMP-7 gene and perform qualitative and quantitative evaluations for mRNA transcription and for Type II collagen and aggrecans protein production. Results will be compared to controls and to adenoviral transfection.

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First published: 07/15/02 Last updated: 11/24/09 webmaster@ortho.wisc.edu
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