Janik Adriaansen

Enhanced gene transfer to arthritic joints using adeno-associated virus type 5: implications for intra-articular gene therapy

Background: Gene therapy of the joint has great potential as a new therapeutic approach for the treatment of rheumatoid arthritis (RA). The vector chosen is of crucial importance for clinical success. Objective: To investigate the tropism and transduction efficiency in arthritic joints in vivo, and in synovial cells in vitro, using five different serotypes of recombinant adeno-associated virus (rAAV) encoding β-galactosidase or green fluorescent protein genes. Methods: rAAV was injected into the ankle joints of rats with adjuvant arthritis after the onset of disease. Synovial tissue was examined at different time points for β-galactosidase protein and gene expression by in situ staining and polymerase chain reaction (PCR) analysis, respectively. In addition, the ability of rAAV to transduce primary human fibroblast-like synoviocytes from patients with RA was investigated in vitro. Results: Intra-articular injection of the rAAV5 serotype resulted in the highest synovial transduction, followed by much lower expression using rAAV2. Expression of the transgene was already detectable 7 days after injection and lasted for at least 4 weeks. Only background staining was seen for serotypes 1, 3, and 4. Importantly, there was a minimal humoral immune response to rAAV5 compared with rAAV2. Additionally, it was found that both rAAV2 and rAAV5 can efficiently transduce human fibroblast-like synoviocytes obtained from patients with RA. Conclusion: Intra-articular rAAV mediated gene therapy in RA might be improved by using rAAV5 rather than other serotypes.

Inflammation‐inducible anti‐TNF gene expression mediated by intra‐articular injection of serotype 5 adeno‐associated virus reduces arthritis

The tumor necrosis factor (TNF)‐α plays a central role in rheumatoid arthritis (RA) and current biotherapies targeting TNF‐α have a major impact on RA treatment. The long‐term safety concerns associated with the repetitive TNF blockade prompt optimization of therapeutic anti‐TNF approaches. Since we recently demonstrated that intra‐articular gene transfer using a recombinant adeno‐associated virus serotype 5 (rAAV5) efficiently transduces arthritic joints, we evaluate its effect on collagen‐induced arthritis (CIA) when encoding TNF antagonists.

Reduction of arthritis following intra-articular administration of an adeno-associated virus serotype 5 expressing a disease-inducible TNF-blocking agent

Background: In the context of preclinical development, we studied the potential of intra-articular gene delivery using a recombinant adeno-associated virus 5 (rAAV5) encoding a chimeric human tumour necrosis factorα (TNFα) soluble receptor I linked to a mouse immunoglobulin heavy chain Fc portion (TNF receptor I; TNFRI-Ig). Methods: Expression was under control of a nuclear factor kappa B (NFκB)-responsive promoter and compared with a cytomegalovirus (CMV) promoter (rAAV5.NFκB-TNFRI-Ig and rAAV5.CMV-TNFRI-Ig, respectively). Results: Fibroblast-like synoviocytes transduced in vitro with rAAV5.NFκB-TNFRI-Ig were able to produce TNFRI-Ig protein in response to several stimuli, and this was inhibited upon treatment with a specific NFκB blocking agent. A bioassay revealed that the synthesised TNFRI-Ig was bioactive, showing a higher affinity for human than for rat TNFα. Transcription of the transgene and protein production were detectable in joints injected with both constructs. No dissemination of the vector was observed outside the joints. A significant reduction in paw swelling was seen in rats treated with rAAV5.NFκB-TNFRI-Ig. This clinical effect was accompanied by a decrease in pro-inflammatory cytokine levels and an increase in IL10 expression in the synovium. Conclusion: These results provide evidence that intra-articular gene therapy using rAAV5 encoding TNFRI-Ig may be a safe and feasible approach for the treatment of rheumatoid arthritis. The higher affinity for human TNFα suggests that in patients with rheumatoid arthritis the therapeutic effect might be even more pronounced than in rat adjuvant arthritis.

CD97 neutralisation increases resistance to collagen-induced arthritis in mice

Synovial tissue of rheumatoid arthritis (RA) patients is characterised by an influx and retention of CD97-positive inflammatory cells. The ligands of CD97, CD55, chondroitin sulfate B, and α5β1 (very late antigen [VLA]-5) are expressed abundantly in the synovial tissue predominantly on fibroblast-like synoviocytes, endothelium, and extracellular matrix. Based upon this expression pattern, we hypothesise CD97 expression to result in accumulation of inflammatory cells in the synovial tissue of RA patients. To determine the therapeutic effect of blocking CD97 in an animal model of RA, collagen-induced arthritis was induced in a total of 124 DBA/J1 mice. Treatment was started on day 21 (early disease) or on day 35 (longstanding disease) with the blocking hamster anti-mouse CD97 monoclonal antibody (mAb) 1B2, control hamster immunoglobulin, or NaCl, applied intraperitoneally three times a week. The paws were evaluated for clinical signs of arthritis and, in addition, examined by radiological and histological analysis. Mice receiving 0.5 mg CD97 mAb starting from day 21 had significantly less arthritis activity and hind paw swelling. Furthermore, joint damage and inflammation were reduced and granulocyte infiltration was decreased. When treatment was started on day 35, CD97 mAb treatment had similar effects, albeit less pronounced. The results support the notion that CD97 contributes to synovial inflammation and joint destruction in arthritis.

Local delivery of beta interferon using an adeno-associated virus type 5 effectively inhibits adjuvant arthritis in rats

Beta interferon (IFN-β) is a cytokine with potent immunomodulatory properties and has been described as a promising therapeutic molecule for the treatment of rheumatoid arthritis (RA). IFN-β was previously overexpressed intra-articularly using an adenoviral vector in rats with adjuvant arthritis (AA) as a model of RA. This effect was powerful, albeit transient due to the vector chosen. Therefore, in the context of pre-clinical development, a delivery vector optimized for intra-articular gene transfer, recombinant adeno-associated virus type 5 (rAAV5), was selected. To exert an optimal effect, protein production should parallel the course of the disease. For this reason, the gene for IFN-β was placed under the control of an inflammation-responsive [nuclear factor (NF)-κB] promoter. After intra-articular injection of the rAAV5 constructs in rats with AA, local transcription of the transgene and production of the IFN-β protein was found, leading to a pronounced and sustained effect on paw swelling when the expression was under the control of the NF-κB-responsive promoter. Additionally, a significant beneficial effect was observed on proteoglycan depletion and erosions. Thus, intra-articular overexpression of IFN-β using a rAAV5 vector exhibits potential as an innovative therapy for the treatment of RA.

Differential Sorting of Human Parathyroid Hormone After Transduction of Mouse and Rat Salivary Glands

Gene transfer to salivary glands leads to abundant secretion of transgenic protein into either saliva or the bloodstream. This indicates significant clinical potential, depending on the route of sorting. The aim of this study was to probe the sorting characteristics of human parathyroid hormone (hPTH) in two animal models for salivary gland gene transfer. PTH is a key hormone regulating calcium levels in the blood. A recombinant serotype 5 adenoviral vector carrying the hPTH cDNA was administered to the submandibular glands of mice and rats. Two days after delivery, high levels of hPTH were found in the serum of mice, leading to elevated serum calcium levels. Only low amounts of hPTH were found in the saliva. Two days after vector infusion into rats, a massive secretion of hPTH was measured in saliva, with little secretion into serum. Confocal microscopy showed hPTH in the glands, localized basolaterally in mice and apically in rats. Submandibular gland transduction was effective and the produced hPTH was biologically active in vivo. Whereas hPTH sorted toward the basolateral side in mice, in rats hPTH was secreted mainly at the apical side. These results indicate that the interaction between hPTH and the cell sorting machinery is different between mouse and rat salivary glands. Detailed studies in these two species should result in a better understanding of cellular control of transgenic secretory protein sorting in this tissue.

Human Parathyroid Hormone Is Secreted Primarily into the Bloodstream After Rat Parotid Gland Gene Transfer

Hypoparathyroidism is a hormone deficiency syndrome that leads to low blood calcium levels and for which current replacement therapy is inadequate. Gene transfer to salivary glands leads to safe and abundant secretion of therapeutic protein into either saliva or the bloodstream. We previously reported the successful transduction of rat submandibular glands with an adenoviral vector encoding human parathyroid hormone (Ad.hPTH), but unfortunately most of the hPTH was secreted into saliva. Because submandibular and parotid glands are morphologically and functionally different, we hypothesized that hPTH sorting might be different in parotid glands. After 2 days, the pattern of hPTH secretion from transduced parotid glands of intact rats was reversed from that of transduced submandibular glands, that is, most transgenic hPTH was detected in serum (5 × 10(10) viral particles per gland; the saliva-to-serum ratio of total hPTH secreted was 0.04). Vector copies were localized to the targeted parotid glands, with none detected in liver or spleen. Ad.hPTH next was administered to parotid glands of parathyroidectomized rats. Two days after delivery no hPTH was detectable in saliva, but high levels were found in serum, leading to normalization of serum calcium and a significant increase in the urinary phosphorus-to-creatinine ratio. This study demonstrates for the first time differential sorting of transgenic hPTH between submandibular and parotid glands, suggesting that hPTH may be a valuable model protein for understanding the molecular basis of transgenic secretory protein sorting in these exocrine glands. We also show the clinical potential of salivary gland hPTH gene therapy for patients with hypoparathyroidism.

Gene Therapy for Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by persistent joint swelling and progressive destruction of cartilage and bone. The primary manifestations are pain, swelling, and limited joint motility due to inflammation of the synovial membrane. In addition to conventional therapies, biologicals targeting cytokines and their receptors have proven useful as specific therapies for RA. Although these new biologicals have improved treatment to a certain extent and do provide proof of principle for targeted therapies, many patients continue to experience inflammation in one or more joints and repeated injections of the recombinant protein are needed for a long-term therapeutic effect. Gene therapy can provide stable, long-term expression of therapeutic proteins at the site of inflammation and thereby improve the treatment of RA and reduce costs related to the treatment with biologicals. Several gene therapy approaches have been developed and tested in animal models of arthritis. Currently a large number of ongoing studies are attempting to improve the efficacy and safety of vectors that are promising for gene therapeutic applications in humans. In addition, studies have been initiated to select new therapeutic candidate genes for the treatment of RA. After almost 20 years of preclinical research the first clinical trials in RA patients have been performed or are ongoing. This review describes the current status of the most promising vectors and therapeutic genes for gene therapy in RA.