Abstract
BackgroundTherapeutic gene transfer is of significant value to elaborate efficient, durable treatments against human osteoarthritis (OA), a slow, progressive, and irreversible disorder for which there is no cure to date.MethodsHere, we directly applied a recombinant adeno-associated virus (rAAV) vector carrying a human transforming growth factor beta (TGF-β) gene sequence to primary human normal and OA chondrocytes in vitro and cartilage explants in situ to monitor the stability of transgene expression and the effects of the candidate pleiotropic factor upon the regenerative cellular activities over time.ResultsEfficient, prolonged expression of TGF-β achieved via rAAV gene transfer enhanced both the proliferative, survival, and anabolic activities of cells over extended periods of time in all the systems evaluated (at least for 21 days in vitro and for up to 90 days in situ) compared with control (reporter) vector delivery, especially in situ where rAAV-hTGF-β allowed for a durable remodeling of OA cartilage. Notably, sustained rAAV production of TGF-β in OA cartilage advantageously reduced the expression of key OA-associated markers of chondrocyte hypertrophic and terminal differentiation (type-X collagen, MMP-13, PTHrP, β-catenin) while increasing that of protective TIMPs and of the TGF-β receptor I in a manner that restored a favorable ALK1/ALK5 balance. Of note, the levels of activities in TGF-β-treated OA cartilage were higher than those of normal cartilage, suggesting that further optimization of the candidate treatment (dose, duration, localization, presence of modulating co-factors) will most likely be necessary to reproduce an original cartilage surface in relevant models of experimental OA in vivo without triggering potentially adverse effects.ConclusionsThe present findings show the ability of rAAV-mediated TGF-β gene transfer to directly remodel human OA cartilage by activating the biological, reparative activities and by regulating hypertrophy and terminal differentiation in damaged chondrocytes as a potential treatment for OA or for other disorders of the cartilage that may require transplantation of engineered cells.
Highlights
Osteoarthritis (OA) is a major, widespread degenerative disease of the entire joint characterized by complex structural and functional tissue and cell alterations [1,2,3,4,5] for which there is no cure to date
These results show that the current recombinant adeno-associated virus (rAAV) TGF-β vector is capable of modifying human normal and OA chondrocytes both in vitro and in situ, allowing for significant levels of transgene expression compared with control vector administration over extended periods of time, especially when the cells are embedded in their extracellular matrix (ECM)
A transferase-mediated dUTP nick end labeling (TUNEL) analysis showed that the presence of TGF-β significantly and durably reduced the percentage of apoptotic cells in OA cartilage compared with lacZ (36-fold decrease, P ≤ 0.001), bringing back the levels to those noted in control normal cartilage (Figures 2B and 4D)
Summary
Osteoarthritis (OA) is a major, widespread degenerative disease of the entire joint characterized by complex structural and functional tissue and cell alterations [1,2,3,4,5] for which there is no cure to date. While protection against cartilage breakdown was afforded by delivering sequences coding for agents with preventive and/or inhibitory activities (an IL-1 receptor antagonist - IL-1Ra, siRNAs against IL-1 or ADAMTS-5, soluble IL-1 and TNF receptors - sIL-1R and sTNFR, NF-κB inhibitors, kallistatin - KBP, thrombospontin-1 - TSP-1, Dickkopf-1 - DKK-1, pro-opiomelanocortin - POMC) [11,12,13,14,15,16,17,18,19,20,21], compensation for the loss of matrix elements and cells was not achieved to further re-establish an original cartilage surface in these various experimental systems Instead, such effects have been ascribed, at least to some extent, to gene transfer of factors with anabolic and/or proliferative properties like proteoglycan 4 [22], the insulin-like growth factor I (IGF-I) [18,23,24], fibroblast growth factor 2 (FGF-2) [25,26], bone morphogenetic proteins 2 and 4 (BMP-2, -4) [23,27], and the transcription factor SOX9 [28,29]. Therapeutic gene transfer is of significant value to elaborate efficient, durable treatments against human osteoarthritis (OA), a slow, progressive, and irreversible disorder for which there is no cure to date
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