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We previously reported that administration of an adeno-associated virus 2 (AAV2) vector encoding a rat tumor necrosis factor (TNF) receptor-immunoglobulin Fc (TNFR:Fc) fusion gene to rats with streptococcal cell wall-induced arthritis resulted in suppression of joint inflammation and cartilage and bone destruction, as well as expression of joint proinflammatory cytokines. In this study, we used an alternate rat model of arthritis to compare the serum levels and duration of TNFR:Fc protein expression following intramuscular administration of pseudotyped AAV-TNFR:Fc vectors based on serotypes 1, 2, and 5. All three pseudotyped AAV-TNFR:Fc vectors led to sustained expression of serum TNFR:Fc protein for at least one year. Serum TNFR:Fc protein levels in rats administered intramuscularly with AAV2/1-TNFR:Fc vector were up to 100- and 10-fold higher than in rats administered the AAV2-TNFR:Fc or AAV2/5-TNFR:Fc vectors, respectively. A single intramuscular administration of AAV2/1-TNFR:Fc vector at vector doses ranging from 10(10) to 10(12) DNase-resistant particles (DRP) per animal, resulted in complete and long-term suppression of recurrent joint inflammation for at least 150 days. Our results establish a proof of concept for administration of an AAV2/1-TNFR:Fc vector to the muscle to achieve long-term, sustained and therapeutically relevant levels of TNFR:Fc protein to treat chronic systemic inflammatory joint diseases.

Top of pageAbstract Rheumatoid arthritis is a chronic autoimmune disease characterized by synovial inflammation, leading to the destruction of cartilage and bone. Previously, we demonstrated that a single intra-articular administration of AAV2-TNFR:Fc vector to rats with experimental arthritis resulted in suppression of disease. For treatment of chronic inflammatory arthritis using intra-articular delivery of AAV2-TNFR:Fc vector, intra-articular re-administrations may be required. In this study, we evaluated if serum anti-AAV2 capsid neutralizing antibodies, elicited after intra-articular administration of AAV2-TNFR:Fc vector to rat joints, affect joint transduction following intra-articular re-administration of an AAV2-FlagLuc vector. AAV2-FlagLuc was administered approximately one, three, nine and twelve months after injection of either vehicle or AAV2-TNFR:Fc vector. Two weeks post intra-articular injection of the AAV2-FlagLuc vector, we measured and compared luciferase enzyme activity in joint tissues of vehicle-treated animals with animals treated first with AAV2-ratTNFR:Fc vector. Intra-articular administration of AAV2-TNFR:Fc vector to rat joints elicited serum anti-AAV2 capsid neutralizing antibody response. Following re-administration with AAV2-FlagLuc, the anti- AAV2 capsid neutralizing antibody levels in the serum were up to 10-fold higher compared with those detected prior to vector re-administration. Luciferase enzyme activity, measured following re- administration of AAV2-FlagLuc vector to vehicle-treated animals, was 25-fold higher compared with the assay background in normal joints, and 12-fold higher in inflamed joints. In contrast, luciferase enzyme activity was not detected in normal or arthritic rat joints that were injected first with AAV2-ratTNFR:Fc and then re-administered with AAV2-FlagLuc. However, using RT-PCR analyses, we observed sustained expression of TNFR:Fc for at least 378 days. These results demonstrate that in rats the levels of serum anti-AAV2 capsid neutralizing antibodies, elicited one to twelve months after AAV2 vector administration to the rat joint, are sufficiently high to inhibit transduction following intra-articular re-administration with a vector of the same serotype. The evidence that TNFR:Fc is expressed in the joints for more than a year suggests that frequent repeat administrations to the joints may not be needed.

TNF-|[alpha]| antagonists such as anti-TNF-|[alpha]| monoclonal antibodies or soluble tumor necrosis factor receptor have proven to be useful therapeutics for the treatment of autoimmune inflammatory diseases including rheumatoid and psoriatic arthritis. In the current study, we employed a monoarticular arthritis model in rats that allowed us to evaluate the long-term effect of TNFR:Fc expression on recurrence of joint inflammation. Monoarticular arthritis was initiated by injection of a small dose of peptidoglycan-polysaccharide polymers (PG-APS) isolated from cell walls of group A streptocci into the hind ankle joint. This resulted in a flare of inflammation that resolved within five days. Intramuscular administration of AAV-TNFR:Fc vectors performed on day seven. To assess the long-term effect of TNFR:Fc expressions on suppression of arthritis in this model, animals were evaluated for four rounds of remission and reactivation of joint inflammation. Reactivation of joint inflammation was induced by intravenous injection of PG-APS. Animals that were treated with vehicle, 109, 5x109 DRP of AAV2/1-TNFR:Fc or 1012 DRP of AAV2-TNFR:Fc developed a significant flare of inflammation in their ankle joints. In these animals, levels of TNFR:Fc protein in the circulation were below 0.75 |[mu]|g/mL. In contrast, when animals were treated with 1010, 1011or 1012 DRP of AAV2/1-TNFR:Fc, levels of TNFR:Fc protein in the circulation were 1.27|[ndash]|8.35 |[mu]|g/mL, 6.8|[ndash]|27.4 |[mu]|g/mL and 19.6|[ndash]|61 |[mu]|g/mL, respectively. The inflammatory response in these animals, as measured by joint volume, was completely suppressed. To increase the transduction efficiency of AAV2/2- TNFR:Fc following administration of 1012 DRP, we employed treatment with proteosome inhibitor that has been shown to enhance AAV transduction. In these animals, levels of TNFR:Fc protein in the circulation increased up to 3.65 |[mu]|g/mL, and led to significant decrease in joint inflammation.

The Ad/AAV hybrid vector is an E1 gene-deleted adenovirus containing a transgene cassette flanked on its 5' and 3' ends by AAV ITR vector sequences. After coinfection of Ad/AAV and wt Ad5 of cells containing the rep-cap genes, the AAV ITR-containing transgene cassette is excised from the Ad/AAV, amplified, and then packaged into AAV particles.

Although periodontitis is initiated by specific oral pathogens that colonize and invade the oral tissues, the host inflammatory response is a major factor in disease progression. Soluble protein delivery of antagonists to tumor necrosis factor alpha (TNF-receptor:Fc fusion protein) inhibit alveolar bone resorption. However, the clinical use of recombinant p75-TNFR:Fc fusion protein raises several concerns, such as reoccurrence of disease activity after cessation of therapy. Objectives: This pilot study used adeno-associated virus (AAV) as a mode to deliver the TNFR:Fc transgene to periodontal lesions in an experimental model of P. gingivalis LPS-mediated bone loss. Methods: Three groups of Sprague-Dawley rats received one of three treatments: LPS delivered to the maxillary interdental gingivae thrice weekly for 8 weeks; vehicle alone (PBS) or LPS plus the single local delivery of pseudotyped AAV[2/5]-TNFR:Fc vector [6 sites|[times]|15uL of 8|[times]|1011 DNase I-resistant particles (DRP)] at baseline. Microcomputed tomography (MicroCT), systemic blood levels of TNFR:Fc protein and cytokine profiles were performed at various timepoints ranging from baseline to 8 weeks following TNFR:Fc transgene delivery. Results: TNFR protein serum levels were low to undetectable in all groups over the entire observation period indicating that local vector delivery did not result in systemic distribution of the soluble TNFR:Fc protein. Real-time PCR showed higher expression of both TNFR1 and TNFR2 than controls (p

TNF-alpha antagonists such as anti-TNF-alpha monoclonal antibodies or soluble tumor necrosis factor receptors have proven to be useful therapeutics for the treatment of autoimmune inflammatory diseases including rheumatoid and psoriatic arthritis. Recently, we have demonstrated that local (intra-articular) or systemic (intramuscular) administration of an AAV2-ratTNFR:Fc vector, encoding the rat tumor necrosis factor-a receptor: immunoglobulin (IgG1) Fc fusion gene, to rats with experimental arthritis led to suppression of disease as reflected in decreased inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and mRNA expression of joint proinflammatory cytokines. In the current study we employed a monoarticular arthritis model in rats that allow us to evaluate the long-term effect of TNFR:Fc expression on recurrence of joint inflammation. Monoarticular arthritis was initiated by injection of a small dose of peptidoglycan-polysaccharide polymers (PG-APS) isolated from cell walls of group A streptocci into the hind ankle joints. This resulted in a flare of inflammation that resolved within 5 to 7 days. Thirty-two days later, reactivation of joint inflammation was induced only in the joints previously injured by intravenous injection of subarthropathic dose of PG-APS. Twenty-six days later, rats were administered intramuscularly with either vehicle or with AAV-TNFR:Fc vector pseudotyped with AAV type 1 capsid at a dose of 1E12 DNase-resistant particles (DRP). Four weeks post-vector administration, the mean levels of circulating TNFR:Fc protein was 21.5 μg/mL. PG-APS was then administered intravenously to induce a recurring flare of joint inflammation. As expected, animals that were treated with vehicle developed a significant flare of inflammation in their ankle joints. In contrast, the inflammatory response, measured by joint volume, was completely suppressed in AAV2/1-TNFR:Fc-treated animals. To assess the long-term effect of TNFR:Fc expression on suppression of arthritis in this model, animals are currently being evaluated for another round of remission and reactivation of joint inflammation.

Recently, we have demonstrated that local (intraarticular) or systemic (intramuscular) administration of an AAV-ratTNFR:Fc vector, encoding the rat tumor necrosis factor-a receptor: immunoglobulin (IgG1) Fc fusion gene, to rats with experimental arthritis led to suppression of disease as reflected in decreased inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and mRNA expression of joint proinflammatory cytokines. Last year we began to evaluate the serum expression levels, in rats, of TNFR:Fc fusion protein following either endotracheal, intramuscular or intravenous administration of AAV-ratTNFR:Fc vectors pseudotyped with capsids from AAV serotypes 1, 2 or 5. Intramuscular delivery proved to be the most efficient route of administration and resulted in very high and sustained serum levels of TNFR:Fc protein for over a year. AAV2/1 was 100- to 1000-fold more efficient than AAV2/5 and AAV2/2, while AAV2/5 was up to 5-fold more efficient than AAV2/2. All animals developed anti-capsid neutralizing antibodies. For AAV2/1, these neutralizing antibodies reduced but not eliminated transduction following repeat intramuscular administration. Following a single intravenous administration, AAV2/1 demonstrated an over a 100-fold enhanced expression compared with AAV2/5 and AAV2/2, and AAV2/5 was up to 10-fold more efficient than AAV2/2. All animals developed anti-capsid neutralizing antibodies. Biodistribution of AAV2/1 vector DNA to perfused organs as well as TNFR:Fc mRNA expression is currently being assessed and data will be presented. Following a single pulmonary delivery of AAV2/5, maximum expression was achieved at 6-weeks post-administration, then gradually declined but remained detectable over a period of more than 250 days. Anti-AAV type 5 capsid neutralizing antibodies were elicited but did not prevent transduction and renewed expression of circulating TNFR:Fc protein for additional 6 months following a second AAV2/5 administration. The serum levels of TNFR:Fc protein were below the limit of detection (< 2 ng/mL) over 9-weeks post-AAV2/2 administration to the lung, although anti-AAV2 neutralizing antibodies were clearly evident. These had no apparent impact on transduction following repeat administration with an AAV2/5 vector. After more than 400 days, AAV2/5-transduced lungs demonstrated 100-fold more TNFR:Fc mRNA than animals administered with AAV2/2. AAV2/1 administration to the lung resulted in secretion of low but detectable TNFR:Fc protein that was almost 10-fold less efficient than AAV2/5. AAV2/1-treated animals developed anti-AAV type 1 capsid neutralizing antibodies. Re-administration of AAV2/5 to AAV2/1-treated animals resulted in renewed high levels of circulating TNFR:Fc protein which declined over a period of 6 months. These results demonstrate the utility of AAV vectors of alternate serotypes for long-term systemic delivery of secreted soluble TNFR:Fc protein and highlight their potential to treat systemic TNF-alpha-mediated autoimmune diseases such as reumatoid arthritis and psoriasis.