Bone marrow-derived macrophages incorporate into the endothelium and influence vascular and renal function after irradiation

IP2Immunopathology of vascular and renal diseases and of organ and celltransplantatio

Human IFN-β expression levels in RA, rodent, rabbit and NHP FLS.

<p>Comparison of hIFN-β expression levels showed that DOX addition 4 hours post-transduction (‘Dox post’) improved expression in RA FLS more than 4-fold compared to DOX treatment 24 hours pre-transduction (‘Dox pre’) and almost 20-fold compared to medium only. In all three conditions, cells were stimulated with TNF-α (A). Transduction with ART-I02 resulted in increased levels of hIFN-β after stimulation of RA FLS. Without addition of ART-I02 or without stimulation (TNF-α and/or IL1-β), no hIFN-β was detected (B). Double stimulation with TNF-α and IL-1β did not increase the level of transgene expression compared to TNF-α stimulation alone (B). Human IFN-β production was detectable in culture supernatants of FLS of all species (C). For panel B and C, in all conditions doxorubicin was used. Data shown are mean + SEM, N = 2–3 experiments.</p

Effect of MTX on transgene expression and bioactivity in RA FLS.

<p>In the presence of MTX hIFN-β transgene expression (A) and bioactivity shown by change in IL-1ra (B) and IL-8 (C) remained unaltered in RA FLS. All samples were stimulated with TNF-α. Control cells were stimulated but not transduced with ART-I02. Data shown are mean + SEM (hIFN-β) and mean + SD (cytokines), N = 3–6 experiments, * indicates P<0.05, ** P<0.01 and *** P<0.001.</p

Vector biodistribution of ART-I02 in arthritic animals.

<p>Vectors were injected intra-articularly (IA) on day 14 after arthritis induction and vector DNA biodistribution was determined 1 and 4 weeks after administration by RT-PCR of a number of tissues (injected joint (right), non-injected joint (left), draining lymphnode, liver, lung, heart, testis, kidney, brain, spleen and blood) (N = 6 per group). Minimal vector spreading outside the joints was detected 1 and 4 weeks after intra-articular administration in animals with (A and B respectively) and without arthritis (C and D respectively). A different pattern was observed after intravenous (IV) administration of the vector, with almost 40% detected in the lungs (E). Data are shown as percentages of the total amount of vector retrieved. CIA, collagen induced arthritis.</p

Vector copies of ART-I02 detected per tissue after intra-articular or intravenous administration.

<p>Besides in the intra-articularly injected right joint, high copy numbers were observed in draining lymph nodes and lung tissue. Intermediate numbers of vector copies were detected in spleen, kidney and blood, low numbers in liver, blood and brain. Levels in heart and testis were very low or below the detection limit of the assay. Data shown are mean + SD, graphs are shown with logarithmic scale. The 100 vg copies detection limit is depicted with a gray dashed line. N = 6 per group. GFP, green fluorescent protein; IA, intra-articular; IV, intravenous; week 1 or 4, number of weeks after vector administration; AA, adjuvant arthritis model.</p

Bioactivity of ART-I02 in human and NHP FLS.

<p>In the presence of hIFN-β the anti-inflammatory cytokine IL-1ra was up regulated in RA FLS (A). Levels of pro-inflammatory cytokines (IL-6, IL-8) as well as MMP-3 were downregulated in RA FLS (A). A quantitative gene reporter bioassay confirmed the presence of bioactive hIFN-β after transduction of ART-I02 in combination with stimulation both in RA FLS as well as NHP FLS (B). In NHP FLS (C), IL-8 was also downregulated significantly, although IL-6 in NHP FLS showed only a trend towards downregulation. All samples were stimulated with TNF-α, except for the human IL-6 data, where samples were stimulated with TNF-α and IL-1β. Control cells were stimulated but not transduced with ART-I02. Data shown are mean + SD, N = 2–6 experiments, * indicates P<0.05, ** P<0.01 and *** P<0.001.</p

Luminescence of rAAV5-CMV-Fluc detected during longitudinal follow up after intra-articular administration.

<p>Expression was monitored for a total of 7 weeks. Expression is visible as early as the first imaging moment (day 3) and remained stable up till day 49 (A). Images over time of 2 representative animals are shown for day 3, 14 and 49 (end of experiment)(B). Data are shown as mean + SEM, N = 5.</p

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

International audienceIntroductionProof 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).MethodsThe 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.ResultsTransduction 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.ConclusionsThese 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