Caroline J. Aalbers

Advancements in adeno-associated viral gene therapy approaches: exploring a new horizon.

Gene therapy is a promising new therapeutic strategy that has been explored in a wide variety of diseases, ranging from cancer to hemophilia, and ocular disorders to autoimmune diseases, among others. Proof of concept of gene transfer approaches has been shown in over 100 studies of animal models of disease, although only a few are under development for clinical application. The US Food and Drug Administration and the European Medicines Agency have not approved any viral human gene therapy products for sale so far, but the amount of gene-related research and development occurring in the United States and Europe continues to grow at a fast rate. This review summarizes the current status of developments in the field of viral gene therapy using adeno-associated virus as a vector, with a special focus on arthritis. For rheumatoid arthritis, and to a lesser extent for other immune-related inflammatory disorders, several cell and gene transfer approaches have been investigated at the preclinical level and a few have been implemented in clinical trials. Finally, both the potential and the hurdles that are faced during development of a viral gene therapy through to its clinical application are discussed.

Preclinical Potency and Biodistribution Studies of an AAV 5 Vector Expressing Human Interferon-β (ART-I02) for Local Treatment of Patients with Rheumatoid Arthritis.

Introduction Proof of concept for local gene therapy for the treatment of arthritis with immunomodulatory cytokine interferon beta (IFN-β) has shown promising results in animal models of rheumatoid arthritis (RA). For the treatment of RA patients, we engineered a recombinant adeno-associated serotype 5 vector (rAAV5) encoding human (h)IFN-β under control of a nuclear factor κB promoter (ART-I02). Methods The potency of ART-I02 in vitro as well as biodistribution in vivo in arthritic animals was evaluated to characterize the vector prior to clinical application. ART-I02 expression and bioactivity after transduction was evaluated in fibroblast-like synoviocytes (FLS) from different species. Biodistribution of the vector after local injection was assessed in a rat adjuvant arthritis model through qPCR analysis of vector DNA. In vivo imaging was used to investigate transgene expression and kinetics in a mouse collagen induced arthritis model. Results Transduction of RA FLS in vitro with ART-I02 resulted in high expression levels of bioactive hIFN-β. Transduction of FLS from rhesus monkeys, rodents and rabbits with ART-I02 showed high transgene expression, and hIFN-β proved bioactive in FLS from rhesus monkeys. Transgene expression and bioactivity in RA FLS were unaltered in the presence of methotrexate. In vivo, vector biodistribution analysis in rats after intra-articular injection of ART-I02 demonstrated that the majority of vector DNA remained in the joint (>93%). In vivo imaging in mice confirmed local expression of rAAV5 in the knee joint region and demonstrated rapid detectable and sustained expression up until 7 weeks. Conclusions These data show that hIFN-β produced by RA FLS transduced with ART-I02 is bioactive and that intra-articular delivery of rAAV5 drives expression of a therapeutic transgene in the joint, with only limited biodistribution of vector DNA to other tissues, supporting progress towards a phase 1 clinical trial for the local treatment of arthritis in patients with RA.

Safety, Biodistribution, and Efficacy of an AAV-5 Vector Encoding Human Interferon-Beta (ART-I02) Delivered via Intra-Articular Injection in Rhesus Monkeys with Collagen-Induced Arthritis

Preclinical studies to assess biodistribution, safety, and initial efficacy of ART-I02, an adeno-associated type 5 (rAAV5) vector expressing human interferon β (hIFN-β), were performed in a total of 24 rhesus monkeys with collagen-induced arthritis. All monkeys were naïve or showed limited neutralizing antibody (Nab) titers to AAV5 at the start of the study. Animals were injected with a single intra-articular dose of ART-I02 or placebo, consisting of 3.2×10(13) vg (Dose A=maximum feasible dose), 4.58×10(12) vg (Dose B), or placebo in the first affected finger joint, the ipsilateral knee, and ankle joint at the same time point. Animals were monitored for clinical parameters and well-being with a maximum of 4 weeks, with the option that the severity of arthritis could necessitate an earlier time point of sacrifice. No adverse events were noted after injection of ART-I02. No abnormalities were observed after histological evaluation of all organs. At both dose levels, immunohistochemical staining indicated expression of hIFN-β. In animals injected with Dose A, we observed stabilization or a reduction in swelling in the finger joint in which vector was administered. The highest copy numbers of vector DNA were detected in synovial tissue of the injected joint and the draining lymph node of the injected knee. High titers of Nab to rAAV5 were observed at the end of the study. Five monkeys developed an rAAV5-specific T-cell response. Two monkeys developed Nab to hIFN-β. In conclusion, intra-articular injection of ART-I02 was well-tolerated and did not induce adverse events. After administration of Dose A of ART-I02, we observed a beneficial effect on joint swelling, substantiated by decreased histological inflammation and bone erosion scores. A GMP vector for clinical application has been manufactured and is currently being tested in GLP rodent studies, with the aim to move forward to a clinical trial.

Intra-articular etanercept treatment in inflammatory arthritis: A randomized double-blind placebo-controlled proof of mechanism clinical trial validating TNF as a potential therapeutic target for local treatment

OBJECTIVE There is an increased interest in developing gene therapy approaches for local delivery of therapeutic genes in patients with arthritis. Intra-articular (i.a.) gene delivery, using an adeno-associated virus encoding a TNF soluble receptor, resulted in reduced paw swelling in an arthritis animal model, but i.a. treatment with a similar vector did not induce robust clinical improvement in patients. It is unclear whether this can be explained by for instance insufficient transduction efficiency or the fact that TNF is not a good therapeutic target for i.a treatment. The objective of this study was to explore the effects of i.a TNF blockade. METHODS Thirty-one patients with rheumatoid or psoriatic arthritis were assigned to a single i.a. injection of 25mg etanercept or placebo in a double-blind randomised controlled clinical trial. The primary end point was target joint improvement, determined by a composite change index. RESULTS Twenty-two patients received etanercept and 9 received placebo. Treatment was generally well tolerated. Treatment with etanercept resulted in a prompt and statistically significant improvement of the index (P<0.001) in comparison with placebo. As expected in light of the half-life of etanercept, the beneficial effect was transient and only statistically significant at week 1 and 2 after i.a. injection. CONCLUSION The results support the development of novel approaches for long-term inhibition of TNF at the site of inflammation, such as gene therapy. TRIAL REGISTRATION The Netherlands National Trial Register (NTR), www.trialregister.nl, NTR-1210.

More outreach for young scientists

Initiatives such as the European Union’s Science in Society programme (see go.nature.com/ MZdJua) spend millions on projects that aim to bring the scientific community closer to the public and the media. We believe it should be easier for young scientists in particular to contribute to such efforts. To this end, we organized a symposium (‘Scientist! Come out of your lab!’) in April 2010. Disclose all data in publications

Advances in local targeted gene therapy for arthritis: towards clinical reality

Margriet J Vervoordeldonk, Caroline J Aalbers & Paul P Tak† †Author for correspondence Academic Medical Center/University of Amsterdam, Division of Clinical Immunology and Rheumatology, F4–218, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands Tel.: +31 205 662 171; Fax: +31 206 919 658; p.p.tak@amc.uva.nl and, Arthrogen BV, Amsterdam, The Netherlands ‘[There is] a strong rationale for IFN-β gene therapy as a novel therapeutic approach for arthritis.’

Single-joint Assessment for the Evaluation of Intraarticular Treatment: Responsiveness and Discrimination of the Composite Change Index

Objective. To investigate responsiveness, discrimination, and construct validity of a composite change index (CCI) for the assessment of single-joint involvement in inflammatory arthritis. Methods. Evaluation of standardized response means (SRM), Guyatt effect size, and Spearman rank correlation coefficient in a randomized controlled trial investigating the effect of an intraarticular etanercept injection. Results. The CCI showed a high SRM (1.68) and high Guyatt effect size (2.72). Both visual analog scale of pain and functionality had a moderate Guyatt effect size (2.06, 2.44) and high SRM (0.81, 0.97). Conclusion. This study supports the use of the CCI as a single-joint assessment after single-joint intervention. Clinical trial registration: NTR-1210.

645. Improving RAAV Gene Expression Inflammatory Conditions Using Empty Capsids and an Immunosuppressive Compound

Objective: Gene therapy has the potential to treat rheumatic diseases. We have shown that rAAV5 is effective in delivering genes to the joint in vivo, however, the presence of macrophages in the inflamed joint might hampering AAV mediated gene delivery. To investigate this further, we determined if administration of agents that influence macrophage activity/number and/or addition of empty capsids had an effect on rAAV5-transgene expression in the joints of arthritic and healthy mice.Material and Methods: Expression of luciferase after i.a. injection of an rAAV5 vector expressing Fire-fly luciferase was investigated in mice with or without arthritis. Mice with collagen-induced arthritis (CIA) were injected with rAAV5.CMV.Fluc or NaCl in both knee joints on day 21 (unless indicated otherwise) and monitored for luciferase expression (from 3 days up till 6 months) by IVIS. Where indicated, empty capsids were co-administered with full particles at a 5:1 or 20:1 (empty: full) ratio. Macrophages were depleted or inhibited by administration of systemic clodronate liposomes (i.v.) or triamcinolone (i.m. or i.a.) 48 hrs prior to vector administration. Healthy mice were administered with vector and empty capsid.Results: We showed that in arthritic mice administration of rAAV5. CMV. Fluc after the onset of inflammation (d24) resulted in lower expression of luciferase compared to vector administration before onset of inflammation (d17). Both the addition of triamcinolone and clodronate improved luciferase expression over a period of 4 weeks (end of experiment). When empty capsids were added to full genome containing capsids in a 1:5 ratio, expression per joint, and the percentage of knee joints with a positive signal improved. A study to assess the duration of expression as well as to investigate the combination of these two approaches was performed. Similar to the previous study, we observed increased expression using triamcinolone or empty capsid alone, however the combination of macrophage inhibition and decoy capsids resulted in a synergistic enhancement of gene expression (5.85 fold, p = 0.001). This increase was sustained for 6 months post vector administration. As triamcinolone is usually administered directly into the inflamed joint of RA patients, we compared the efficacy of i.a. versus i.m. administration of triamcinolone and found that the enhancement of gene expression was independent of the route of administration. Next we wanted to see if empty capsid could enhance expression in non-inflamed joints. We observed increased expression in the presence of empty capsid, 4.8 fold at 5:1 ratio and up to 20 fold with the 20:1 ratio (p<0.05). In all experiments expression was limited to the knee joint region.Conclusions: We demonstrated that either depletion or inhibition of macrophages leads to an increase in gene expression. In addition, empty decoy capsids improve transgene expression in the presence or absence of inflammation. These data have implications for future applications of local gene therapy to the joint, or to other tissues that have an abundance of macrophages.

Towards gene therapy for the treatment of rheumatoid arthritis: production and bioactivity of interferon β in fibroblast-like synoviocytes transduced with adeno-associated virus type 5 expressing human interferon β

Background and objectives The authors are developing local intra-articular gene therapy for the treatment of rheumatoid arthritis (RA) using human interferon β (hIFNβ) as therapeutic gene expressed under control of an inflammation-inducible promoter and recombinant adeno-associated virus type 5 as vector (ART-I02). To evaluate hIFNβ expression by ART-I02 and bioactivity, fibroblast-like synoviocytes (FLS) from RA patients and different animal species were transduced with ART-I02 and the culture medium was analysed. Since most RA patients in their future phase I trial will be on methotrexate (MTX) treatment, the authors investigated the influence of MTX on transduction efficacy. Methods Primary human, monkey, mouse and rat and non-primary rabbit FLS were transduced with ART-I02 (MOI 200.000). To activate the nuclear factor-κB promoter, cells were stimulated with tumour necrosis factor (TNF) (1 ng/ml) with or without interleukin 1β (IL-1β) (10 ng/ml) 4 or 24 h post-transduction. In addition, human FLS were incubated in medium with MTX (10 nM, 1 μM, 100 μM) added pretransduction and post-transduction. Supernatants were harvested 48 h after stimulation. Production of hIFNβ was measured by ELISA. Bioactivity was established by analysing the effect on pro-inflammatory cytokine (IL-6, IL-8) and matrix metalloproteinases (MMP) production by ELISA and with a quantitative gene reporter bioassay. Results Human IFNβ production was detectable in supernatants of FLS of all species, with comparable levels in human and monkey FLS. In human FLS transduced with ART-I02, the inhibition of IL-8 (80%) and MMP3 (60%) secretion was most pronounced after stimulation with TNF 24 h after transduction (p<0.05). IL-6 production was significantly (p<0.05) reduced by hIFNβ in cells stimulated with both TNF and IL-1β. Monkey FLS expressing hIFNβ showed a 40% decrease in IL-8 secretion (p<0.01) after stimulation with TNF. No effect of hIFNβ on cytokine and MMP secretion was observed in rabbit and rodent FLS. Human IFNβ produced by both human and monkey FLS showed robust levels of bioactivity in the gene reporter bioassay. MTX treatment did not influence hIFNβ production by ART-I02 or bioactivity. Conclusion Transduction of FLS with ART-I02 resulted in significant hIFNβ expression in FLS of all species. Moreover, hIFNβ produced by ART-I02-transduced cells is bioactive in human and monkey FLS. This study supports the use of non-human primates in a non-clinical pharmacology-toxicity programme and represents a next step towards a phase I clinical trial in RA patients.