Background/Purpose:

Cartilage destruction is accompanied by a tremendous loss of life quality due to deprivation of agility, flexibility and pain. Additionally, increased catabolic mediators such as inflammatory cytokines, matrix-degrading enzymes or bioenergetic-relevant proteins play a crucial role in the pathogenesis of rheumatic disorders such as OA and RA. However, therapeutic approaches including the cartilage restoration or replacement combined with anti-inflammatory properties are still elusive. Therefore, promising approaches link tissue engineering to gene therapy. To overcome critical challenges such as transient therapeutic effects as well as undesirable long-lasting overexpression of transgenes, several approaches focus on the expression control and optimization of viral vectors applied either directly or by a cell therapeutic approach.

Therefore, we aimed at engineering scaffold-free cartilage transplants (SFCTs) using the transgenic expression of the chondroprotective IL-4 under the control of the cyclooxygenase-2 promoter (pCox-2) being activated by the presence of and inactivated in the absence of inflammatory mediators and co-expressing the green fluorescence protein (GFP).

Methods:

First, the gene therapeutic approach (pCox-2 and IL-4) was tested by transient transfection of primary equine chondrocytes, subsequent activation with IL-1β and TNF-α and detection of inflammatory marker and matrix degrading enzyme gene expression (IL1B, TNFA, IL6, IL8, COX2 and MMP1, MMP3). Secondly, the equine and human sequences of pCox-2 and IL-4 were subcloned into a lentiviral-based GFP-co-expressing vector backbone. Thirdly, to assess the durability of GFP-expression SFCTs were generated from primary chondrocytes, transduced and monitored for up to 4 months. Finally, to simulate OA, SFCTs were stimulated with IL-1β and TNF-α for 3 weeks and cultivated afterwards under non-stimulating conditions to determine the regenerative potential. Subsequently SFCTs were analyzed with regard to the inflammatory marker / matrix degrading enzyme gene expression and histological changes (Collagen I/II).

Results:

Feasibility and functionality of the gene therapeutic approach including the genetic switch was demonstrated by IL-4 mediated inactivation of inflammatory marker gene expression and matrix degrading enzyme gene expression. Generated SFCTs achieved diameters up to 1 cm. GFP-expression within transduced SFCTs was stable for up to 4 months. After stimulation of SFCTs with IL-1β and TNF-α, the inflammatory marker gene expression and matrix degrading enzyme gene expression was increased as compared to the untreated controls. The histological findings showed increased softening and wateriness of the tissue. A histological redistribution of Collagen I and II production could be demonstrated. The observed effects were reversible after 3 weeks of regeneration.

Conclusion:

First results show the feasibility and functionality of the gene therapeutic approach, promising features (stability and integrity) of our new generation cartilage transplants combined with gene therapy where the therapeutic gene will be expressed in a disease responsive way. Further investigations are needed to test and optimize the system.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/combining-scaffold-free-cartilage-transplants-to-controlled-gene-expression-for-therapeutic-application-in-rheumatic-disorders/