Volume 3, Issue 6, Pages 901-910 (June 2001) Gene Transfer of p53 to Arthritic Joints Stimulates Synovial Apoptosis and Inhibits Inflammation  Qingping Yao, Sujing Wang, Joseph C. Glorioso, Christopher H. Evans, Paul D. Robbins, Steven C. Ghivizzani, Thomas J. Oligino  Molecular Therapy  Volume 3, Issue 6, Pages 901-910 (June 2001) DOI: 10.1006/mthe.2001.0343 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Adenoviral-mediated transfer of p53 to primary human RA synovial fibroblasts reduces cell viability. Freshly isolated cells were seeded in 24-well plates and at confluence, were either untreated (mock) or infected with the indicated multiplicity of infection (m.o.i.). At 48 h postinfection MTT assays were performed on the cells. Four replicates were performed for each group and the mean taken. In each case, values shown represent cell viabilities relative to mock controls. Error bars reflect ±SD. Molecular Therapy 2001 3, 901-910DOI: (10.1006/mthe.2001.0343) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Adenoviral-mediated overexpression of p53 affects the viability of synovial fibroblasts isolated from both RA and OA patients. Freshly isolated cells were seeded in 24-well plates and at confluence were either untreated (mock) or infected with Ad.lacZ or Ad.p53 (m.o.i. 100). Forty-eight hours postinfection, MTT assays were performed on the treated cells. Four replicates were performed for each group and the mean taken. In each case, values shown represent cell viabilities relative to mock controls. Each cell line was compared against itself. Error bars reflect ±SD. Molecular Therapy 2001 3, 901-910DOI: (10.1006/mthe.2001.0343) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Adenoviral-mediated overexpression of p53 in primary rabbit synovial fibroblasts reduces cell viability. Rabbit synovial fibroblasts isolated from both arthritic and normal rabbit knees were infected with the indicated vector at an m.o.i. of 100. Forty-eight hours postinfection MTT assays were performed on the cells. Four replicates were performed for each group and the mean taken. Values shown represent cell viabilities relative to mock controls. Error bars reflect ±SD. Molecular Therapy 2001 3, 901-910DOI: (10.1006/mthe.2001.0343) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Ad.p53 treatment significantly reduces inflammation in arthritic joints. Three days after the induction of arthritis in rabbit knees both knees were treated with either saline or 1 × 1011 particles of the indicated vector. Prior to vector treatment, the joint space of each knee was lavaged with 1 ml saline and the number of WBC/ml synovial fluid determined (Day 0). Groups of animals were sacrificed at periodic intervals after vector treatment and WBC/ml synovial fluid determined as above. At each time point inflammation is expressed as a percentage relative to the inflammation observed in saline-treated joints which were assigned a value of 100%. Error bars reflect ±SD. Molecular Therapy 2001 3, 901-910DOI: (10.1006/mthe.2001.0343) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 Massive apoptosis can be detected in p53-treated synovium 24 h after vector delivery. Serial sections taken from joints treated with 0.25 ml saline containing 1 × 1011 particles of either Ad.lacZ or Ad.p53 were stained with hematoxylin and eosin (A–D) or tested for apoptosis by TUNEL staining (E and F). Sections taken from Ad.lacZ-treated joints (A, C, E) displayed little or no apoptosis. In contrast sections taken from p53-treated joints displayed large regions of tissue consistent with apoptosis when stained with H & E (B and D). These regions were also highly positive when subjected to TUNEL staining (F). Molecular Therapy 2001 3, 901-910DOI: (10.1006/mthe.2001.0343) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 6 Intraarticular delivery of Ad.p53 significantly reduces the cellularity of the synovial lining. Three days after the induction of arthritis in rabbit knee joints animals were injected intraarticularly in both knees with 0.25 ml saline or saline containing 1 × 1011 particles of Ad.lacZ or Ad.p53. Four days after vector delivery animals were sacrificed and synovial tissue harvested. Tissue was fixed and serial paraffin-embedded sections were taken and stained with hematoxylin and eosin (H & E). Sections from joints treated with Ad.lacZ (A) were indistinguishable from buffer controls. Ad.lacZ-treated sections displayed dramatically thickened synovium with a large number of infiltrating cells present throughout the tissue. In contrast, sections from joints treated with Ad.p53 (B) displayed large regions of synovium that were essentially devoid of cells and consisted mainly of fibrous matrix, indicating that the cells normally found in this tissue had been eliminated. Molecular Therapy 2001 3, 901-910DOI: (10.1006/mthe.2001.0343) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 7 Intraarticular delivery of adenoviral vectors mediates transgene expression only in the cells proximal to the joint space. Arthritic rabbit knees were injected with either saline (A) or saline containing 1011 particles of an adenoviral vector expressing the human alkaline phosphatase gene (B). Forty-eight hours after delivery of the vectors animals were sacrificed and the synovium harvested. Harvested tissue was snap frozen on dry ice and 20-μm sections were taken. Sections were then assayed for alkaline phosphatase activity as described under Materials and Methods. Molecular Therapy 2001 3, 901-910DOI: (10.1006/mthe.2001.0343) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 8 Intraarticular delivery of Ad.p53 does not inhibit GAG synthesis in articular cartilage. Rabbit knees were treated with 1 × 1011 particles of Ad.lacZ or Ad.p53 in 0.25 ml saline 4 days after the induction of arthritis. Ten days after vector delivery, animals were sacrificed and cartilage shavings isolated from the femoral condyles. Isolated cartilage was incubated with 35SO4−2 for 24 h and the incorporation of label into proteoglycans determined. Molecular Therapy 2001 3, 901-910DOI: (10.1006/mthe.2001.0343) Copyright © 2001 American Society for Gene Therapy Terms and Conditions