Sustained Interleukin-10 Transgene Expression Following Intra-articular AAV5-IL-10 Administration in Horses

Presented by: Kyla Ortved

 

 Authors: Kyla F. Ortved, DVM, PhD, DACVS, DACVSMR; Kaitlyn L. Moss, BA, MAT; Zibin Jiang, PhD; Michael E. Dodson, BA, MBs; Renata L. Linardi, DVM, PhD; Joanne Haughan, Mag. Med. Vet.; Alexis L. Gale, BSc; Cara Grzybowski, BSc; Julie E. Engiles, VMD, DACVP; Mary A. Robinson, VMD, PhD, DACVCP

Affiliations: Department of Clinical Studies, New Bolton Center, University of Pennsylvania

Introduction: Joint trauma leads to post-traumatic inflammation with upregulation of inflammatory cytokines and degradative enzymes. If severe enough, this response can lead to irreversible post-traumatic osteoarthritis (PTOA). A regenerative therapeutic that mitigates inflammation in the posttraumatic joint would be greatly beneficial. Interleukin-10 (IL-10) has potent anti-inflammatory effects and has protective effects in inflamed equine chondrocytes. A gene therapy approach using a vector to overexpress IL-10 in the joint represents a feasible method of delivering sustained, high doses of IL-10 in the posttraumatic joint. Adeno-associated virus (AAV) is non-pathogenic viral vector that represents an ideal gene therapy vector for intraarticular therapy.

Hypothesis / Objectives: The objectives were to investigate the intraarticular response to vector injection including inflammation and transgene production, and to investigate systemic distribution of transgene and vector. We hypothesized that AAV5-IL-10 injection would result in sustained IL-10 expression without inducing intrasynovial inflammation and that injection that would not affect systemic IL-10.

Materials and Methods: The middle carpal joint of horses free of carpal OA were injected with AAV5-IL-10 (n=6) or saline (n=6). Synovial fluid (SF) was collected on days 0, 1, 2, 4, 7, 14, 28, 56, and 84. Nucleated cell content (NCC), total protein (TP) and inflammatory cytokines (IL-10, IL-6, IL-17a, IL-1, TNF, and IFN-) were quantified. Serum IL-10 was also quantified. Total SF and serum vector genomes were quantified using qPCR. A mixed effects model was used to analyze continuous data. Statistically significant differences between groups were determined using a Wilcoxon rank sum test. P < 0.05 was considered significant.

Results: Injection with saline and AAV5-IL-10 led to transient increases in NCC and TP (Fig 1). Inflammatory cytokines were not significantly different between groups, except IL-17a (increased on day 2 in AAV5-IL-10 joints), and IL-1 and IL-6 (decreased on day 84 in AAV5-IL-10 joints) (Fig 2). IL-10 was significantly increased in AAV5-IL-10 SF at all time points (Fig 3). Serum IL-10 was not different between groups (Fig 3). Vector genomes were not detectable in SF, serum or synovial fluid in saline injected horses. Vector genomes were detectable in SF, serum and synovial membrane of all AAV5-IL-10 horses (Fig 4, 5).

Conclusions: AAV5-IL-10 injection led to sustained increase in SF IL-10. One major concern with IL-10 overexpression is systemic perturbations, however, no changes in serum IL-10 were noted. Additionally, intraarticular AAV5-IL-10 did not lead to a clinically significant inflammatory response and may actually decrease inflammatory cytokine production over time as IL-1 and IL-6 were lower on day 84. Interleukin-10 is a potent, anti-inflammatory cytokine with potential to modulate articular inflammation, thereby protecting articular cartilage from degradation and PTOA. This study demonstrates the feasibility of intraarticular AAV5-IL-10 and future studies are planned to evaluate IL-10 gene therapy in joints with experimentally-induced PTOA.

Acknowledgements, Funding, and Conflicts of Interest: Funding was provided by The McCabe Fund Advisory Committee. The authors have no conflicts of interest to disclose.

Figures

Figure 1. Synovial fluid analysis following injection. a) NCC was significantly increased over baseline on day 1 following injection in both groups and returns to baseline by day 7 in saline injected horses and day 28 in AAV5-IL-10 injected horses. b) TP was significantly increased over baseline on day 1 following injection in both horses and returns to baseline by day 4 in saline injected horses and day 2 in AAV5-IL-10 injected horses.

Figure 2. Inflammatory cytokine concentration in synovial fluid over time following saline or AAV5-IL-10 injection including a) IL-1, b) IL-6, c) IL-17a, d) TNF- and e) IFN-. *Significant difference between treatment group, p<0.05

Figure 3. Interleukin-10 concentration in a) synovial fluid and b) serum over time. Individual horses are depicted for serum IL-10 analysis due to high inter-animal variability in the saline group.

Figure 4. Copy number of AAV vector genomes in a) synovial fluid and b) serum of individual horses. No detectable vector genomes were detected in any saline injected horse.

Figure 5. a) Copy number of AAV vector genome/g gDNA in synovial membrane collected 84 days post-injection in individual horses. No detectable vector genomes were detected in the synovial membrane from saline-injected horses. b) Box plot of IL-10 mRNA expression in synovial membrane collected 84 days post-injection relative to synovial membrane from saline-injected horses. 18S was used a reference gene in the analysis.