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Blood, Vol. 112, Issue 12, 4532-4541, December 1, 2008
Intraarticular factor IX protein or gene replacement protects against development of hemophilic synovitis in the absence of circulating factor IX
Blood Sun et al.
112: 4532
Supplemental materials for: Sun et al
Files in this Data Supplement:
- Figure S1. AAV-GFP transduction of subpopulations of joint cell types varies with serotype of AAV vector (JPG, 53.6 KB)
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All cell lines and tissue explants were provided generously by the Musculoskeletal Tissue Bank of the UNC-CH Thurston Arthritis Research Center and were collected with informed patient consent following approval by the UNC Internal Review Board for Human Studies. (Upper panels) Primary cell lines of fibroblast-like synoviocytes (FLS) were transduced by self-complementary AAV2, AAV5, or AAV8 vectors encoding GFP at MOI 5000 vg/cell. 5 days later, serial sections were observed for fluorescence. Representative × 200 images shown. (Lower panels) Cartilage from fresh joint tissue explants (~100 mg fragments) obtained from the operating room and maintained in tissue culture were overlaid with 5 × 109 v.g. scAAV.GFP packaged in serotypes 2, 5, or 8 as indicated. 4 days later, serial sections were observed for fluorescence, with representative × 200 images shown. The AAV2 vector directed the strongest GFP expression in FLS, followed by AAV5 vector, with virtually no transduction from AAV8 in vitro. Chondrocyte biology is altered when cells are not studied within the rich extracellular matrix of cartilage that they produce, so cartilage transduction was studied by overlaying AAV-GFP virus on fresh cartilage surgical explants. While most vectors transduced some chondrocytes, AAV8 transduction was most efficient, followed by AAV5 and AAV2. The contrasting tropism of AAV serotypes, even within cell subpopulations of a single tissue, is demonstrated in Fig. 3 by comparing AAV2 (strong GFP gene expression in FLS/weak expression in chondrocytes) versus AAV8 (minimal expression in FLS/strong transduction in chondrocytes).
- Figure S2. hFIX protein and transgene copy analysis after intraarticular delivery of AAV.FIX: AAV2 and AAV5 localize in the joint and while AAV8 protein expression and genome persistence are higher in liver than joint (JPG, 72.3 KB)
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FIX−/− mice received AAV hFIX (2.5 × 109 vg) in left hindlimb knee joint. Four weeks following transduction, liver and injected joint (extraarticular soft tissue removed and discarded) were frozen separately, homogenized in lysis buffer (Promega, Madison, WI) using a polytron, centrifuged, and the supernatant assayed. Total protein concentration was determined with Bradford assay (Pierce, Rockford, IL). (A) hFIX was measured by ELISA,32 and expressed as mean± SEM in ng FIX/mg protein. Factor IX signal in liver from AAV2 and AAV5 is not significantly different from the artifact recorded in untreated joint tissues. (B) hFIX transgene copies in liver and joint homogenate was quantified by FIX Q-PCR,32 and the hFIX cDNA copy numbers expressed as mean ± SEM in genomes/mg protein. The majority of AAV8.hFIX genomes persist in the liver, while spread and persistence of AAV2.hFIX and AAV5.hFIX genomes from joint to liver were not detected.
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