Influence of serotype, cell type, tissue composition, and time after inoculation on gene expression in recombinant adeno-associated viral vector–transduced equine joint tissues

Jeffrey B. Mason Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348.

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Luk H. Vandenberghe Gene Therapy Program, Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.

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Ru Xiao Gene Therapy Program, Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.

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James M. Wilson Gene Therapy Program, Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.

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Dean W. Richardson Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348.

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Abstract

Objective—To evaluate transduction efficiency of gene therapy for treatment of osteoarthritis in horses.

Sample—Cartilage and synovial tissues were aseptically collected from the stifle joints of 3 Thoroughbreds; horses were 3, 7, and 12 years old and free from sepsis and long-term drug treatment and were euthanized for reasons unrelated to joint disease.

Procedures—Gene transfer experiments were performed with 8 recombinant adeno-associated viral vector (rAAV) serotypes in monolayer-cultured equine chondrocytes, synovial cells, and mesenchymal stromal cells and in cartilage and synovial tissues.

Results—Serotypes rAAV2/5 and rAAV2/2 yielded the highest transduction efficiency in cultured cells 6 days after transduction. Synovial cells and mesenchymal stromal cells were more readily transduced than were chondrocytes. Serotype rAAV2/6.2 yielded the highest rate of gene expression in both cartilage and synovial tissues at 6 days after inoculation. However, at 30 and 60 days after inoculation, gene expression of serotypes rAAV2/2 and rAAV2/5 surpassed that of rAAV2/6.2 and all other serotypes.

Conclusions and Clinical Relevance—Maximally expressing serotypes changed between 6 and 30 days in tissues; however, the most efficient serotypes for transduction of joint cells over time were also the most efficient serotypes for transduction of joint tissues. In addition, the low transduction efficiency of articular cartilage tissue was paralleled by a low transduction efficiency of isolated chondrocytes. This suggested that the typically low transduction efficiency of articular cartilage may be attributable in part to the low transduction efficiency of the chondrocytes and not solely a result of the dense cartilage matrix.

Abstract

Objective—To evaluate transduction efficiency of gene therapy for treatment of osteoarthritis in horses.

Sample—Cartilage and synovial tissues were aseptically collected from the stifle joints of 3 Thoroughbreds; horses were 3, 7, and 12 years old and free from sepsis and long-term drug treatment and were euthanized for reasons unrelated to joint disease.

Procedures—Gene transfer experiments were performed with 8 recombinant adeno-associated viral vector (rAAV) serotypes in monolayer-cultured equine chondrocytes, synovial cells, and mesenchymal stromal cells and in cartilage and synovial tissues.

Results—Serotypes rAAV2/5 and rAAV2/2 yielded the highest transduction efficiency in cultured cells 6 days after transduction. Synovial cells and mesenchymal stromal cells were more readily transduced than were chondrocytes. Serotype rAAV2/6.2 yielded the highest rate of gene expression in both cartilage and synovial tissues at 6 days after inoculation. However, at 30 and 60 days after inoculation, gene expression of serotypes rAAV2/2 and rAAV2/5 surpassed that of rAAV2/6.2 and all other serotypes.

Conclusions and Clinical Relevance—Maximally expressing serotypes changed between 6 and 30 days in tissues; however, the most efficient serotypes for transduction of joint cells over time were also the most efficient serotypes for transduction of joint tissues. In addition, the low transduction efficiency of articular cartilage tissue was paralleled by a low transduction efficiency of isolated chondrocytes. This suggested that the typically low transduction efficiency of articular cartilage may be attributable in part to the low transduction efficiency of the chondrocytes and not solely a result of the dense cartilage matrix.

Contributor Notes

Drs. Vandenberghe and Xiao's present address is Berman-Gund Laboratory for Retinal Degenerations, Howe Laboratory, Ocular Genomics Institute, and Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 325 Cambridge St, Boston, MA 02114.

Supported in part by Brushwood Stables; the James Foundation; the United States Equestrian Federation Equine Health Research Fund; the Vector Core of the Gene Therapy Program, University of Pennsylvania; and a grant to Dr. Wilson from the National Institutes of Health (grant No. 2-P30-DK047757-16).

Presented in part at the Annual Experimental Biology Conference, New Orleans, April 2009.

The authors thank Drs. Marty Keough, Julie Johnstone, Peter Bell, and Sadik Kassim and Andrea Phillips for technical assistance.

Address correspondence to Dr. Mason (masonjef@vet.upenn.edu).
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