Document Type

Dissertation

Date of Degree

Summer 2015

Degree Name

PhD (Doctor of Philosophy)

Degree In

Biomedical Engineering

First Advisor

James A. Martin

Abstract

Lubricin, or proteoglycan 4 (PRG4), is a secreted, glycosylated protein that binds to cartilage surfaces, which functions as a boundary lubricant. The loss of lubricin from cartilage is identified as a major pathogenic factor in post-traumatic osteoarthritis (PTOA) that has now been the aim of therapeutic intervention. Intra-articular injection of PRG4 protein provides short-term benefits that might be extended using sustained delivery methods such as in gene therapy.

Here we describe the development and testing of such therapy using adeno-associated virus (AAV) as a vector for the transfer of PRG4-green fluorescent protein (GFP) fusion gene. Our recombinant PRG4 gene produces a PRG4-GFP fusion protein to facilitate tracking of its expression and distribution on joint surfaces. We hypothesized that PRG4-GFP is fully functional as a cartilage lubricant and that PRG4-GFP produced in vivo is expressed by synoviocytes and other joint cells, and cartilage surfaces remained coated for several weeks up to months after intra-articular injection of the virus.

PRG4-GFP showed lubricin-like cartilage binding in vitro, and lubrication immunoblot analysis confirmed that purified PRG4-GFP from cultured media conditioned by PRG4-GFP-transduced synoviocytes was heavily glycosylated, while confocal microscopy revealed binding of the fluorescent fusion protein to cartilage surfaces. Metal-on-cartilage friction tests showed that PRG4-GFP reduced friction coefficients to a degree comparable to that of synovial fluid and had strong chondro-protective effects in explanted cartilage exposed to shear loading. The chondrocyte viability after shear loading showed that PRG4-GFP had a strong chondro-protective effect on par with that of the synovial fluid. Confocal microscopy and immunohistology confirmed that cartilage surfaces in the stifle joints of mice injected with viruses were coated with PRG4-GFP for up to 2 or 4 weeks after the treatment. The overexpression of PRG4-GFP and coating of cartilage surfaces in the stifle joints of mice injected with Adeno-Associated Virus for the transfer of PRG4-GFP fusion gene (AAV-PRG4-GFP) was confirmed by confocal microscopy and immunohistology for up to 2 or 4 weeks post-injection. The μCT imaging and immunohistology in AAV-PRG4-GFP injected rabbit knees showed stronger inhibition in degeneration of damaged tissues than in AAV-GFP injected control group. Collectively these findings indicate that AAV-PRG4-GFP transduction is a valuable new tool for evaluating the effects of long-term lubricant supplementation on PTOA in animal models.

Public Abstract

There are several types of degenerative joint disease in human body joints; rheumatoid arthritis (RA), osteoarthritis (OA), and post-traumatic osteoarthritis (PTOA). This study focused on OA, or PTOA, which is caused by mechanical damage, aging joints, injury, and obesity. OA usually develops in joints that are injured by repeated overuse and exceeded mechanical force such as performing a particular task or playing a favorite sport or carrying around excess body weight.

Over the past few decades, many researchers and scientists introduced a plenty of gene therapy methods such as injection of anti-inflammatory drugs, nutrients, regeneration factors, and lubricants for OA cure and treatment. Nowadays, physical therapy and joint splinting and joint replacement surgery are also developed by a large number of researchers; however, there is neither an ideal treatment of OA, nor is there a complete understanding of OA progression mechanism. Over 27 million in the US population still suffer from OA/PTOA, and a tremendous amount of money and effort are poured into finding the cure for OA/PTOA.

In this study, we focused on development of enhanced gene therapy which uses the recombinant lubricin (PRG4-GFP). Lubricin, also known as proteoglycan 4 (PRG4), is the main lubricant acting as synovial fluid to reduce friction and wear. It is vital to maintain proper function within knee joints.

We developed and tested a gene therapy using adeno-associated virus (AAV) as a vector for the transfer of PRG4-green fluorescent protein (GFP) fusion gene. A friction test with PRG4-GFP showed lubricin-like cartilage binding capacity and inhibiting progression of OA after damage and lubrication. An immunoblot analysis confirmed the production and function of PRG4-GFP. The expression of PRG4-GFP and the coating of cartilage surfaces in the stifle joints of mice/rabbits injected with AAV for the transfer of PRG4-GFP fusion gene (AAV-PRG4-GFP) were confirmed by a confocal microscopy and immunohistology.

Keywords

publicabstract, Friction coefficient, Gene therapy, Green fluorescent protein, Lubricin, Osteoarthritis, PRG4

Pages

xii, 81

Bibliography

72-81

Copyright

Copyright 2015 Hyeong Hun Choe

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