Intra-articular gene therapy blocking NF-κB using adeno-associated virus type 5 ameliorates adjuvant arthritis in rats

Objective

NF-κB is highly activated in synovium of RA patients, and can induce transcription of proinflammatory cytokines, adhesion molecules, and inducible nitric oxide, among others. Phosphorylation of the inhibitor of κB (IκB) proteins is an important step in NF-κB/Rel activation and is regulated by IκB kinase (IKK). The IKK complex consists of at least three subunits, including IKKα and IKKβ (also called IKK1 and IKK2) and the regulatory subunit IKKγ. In an initial study in Lewis rats with adjuvant arthritis (AA) adenoviral dominant-negative IKK2 (Ad.IKK2dn) significantly ameliorated the severity of disease as evidenced by decreased paw swelling compared with Ad.GFP-treated rats [1]. However, adenoviral vectors are known to be very immunogenic, compromising stable long-term expression of the transgene. Adeno-associated virus (AAV) is considered the most promising vector for gene therapy in RA. In a comparative serotype study we found that direct injection of AAV5 into the ankle joints of rats with AA resulted in the highest synovial transduction, with good expression of the transgene at the protein level until the end of the study, followed by AAV2. In the present study we investigated the effect of inhibiting NF-κB in AA in rats using AAV-mediated intra-articular gene therapy. For this purpose we used the following vectors: AAV5 containing the IKK2dn gene (AAV5.IKK2dn) or AAV2 containing the IκBα-supressor gene (AAV2.IκBα SR).

Methods

AAV5.IKK2dn (2.5 × 10¹⁰vp), AAV2.IκBαSR (2.5 × 10¹⁰vp) or AAV5/ AAV2.GFP were injected into the right ankle joints of rats with AA on day 11 after adjuvant immunization. Subsequently, the effect of both genes on paw swelling was measured by water displacement plethysmometry. Animals were sacrificed 2 weeks after intra-articular injection and joints were collected for analysis. Bone degradation was examined using X-rays of the ankle joints and histology was performed to assess synovial inflammation and joint damage.

Results

In the rats treated with IKK2dn, significantly reduced paw swelling was observed (P < 0.05, AAV5.dnIKK2 versus AAV5.GFP). No significant effect was found on cartilage and bone destruction. Intra-articular treatment of the rats with AAV2.IκBα SR only showed a marginal effect on the clinical course of arthritis.

Conclusion

We demonstrate that AAV5-mediated IKK2dn gene transfer to the synovium reduces the severity of inflammation in AA rats, when the treatment was started after the onset of disease. In contrast, injection of AAV2.IκBα SR had a poor clinical effect on paw swelling in rats with AA. This could either be due to the dissimilarities in the mechanisms that these genes use to inhibit NF-κB activation (IKK2 versus IκBα) or to the use of different AAV serotypes as vector (AAV5 versus AAV2).