The authors are investigating self-complementary adeno-associated virus (scAAV) as a vector for intra-articular gene-delivery of interleukin-1 receptor antagonist (IL-1Ra), and its therapeutic capacity in the treatment of osteoarthritis (OA). To model gene transfer on a scale proportional to the human knee, a frequent site of OA incidence, studies were focused on the joints of the equine forelimb. Using AAV2.5 capsid and equine IL-1Ra as a homologous transgene, a functional ceiling dose of similar to 5x10(12) viral genomes was previously identified, which elevated the steady state levels of eqIL-1Ra in synovial fluids by >40-fold over endogenous production for at least 6 months. Here, using an osteochondral fragmentation model of early OA, the functional capacity of scAAV.IL-1Ra gene-delivery was examined in equine joints over a period of 12 weeks. In the disease model, transgenic eqIL-1Ra expression was several fold higher than seen previously in healthy joints, and correlated directly with the severity of joint pathology at the time of treatment. Despite wide variation in expression, the steady-state eqIL-1Ra in synovial fluids exceeded that of IL-1 by >400-fold in all animals, and a consistent treatment effect was observed. This included a 30-40% reduction in lameness and similar to 25% improvement in total joint pathology by both magnetic resonance imaging and arthroscopic assessments, which included reduced joint effusion and synovitis, and improved repair of the osteochondral lesion. No vector-related increase in eqIL-1Ra levels in blood or urine was noted. Cumulatively, these studies in the equine model indicate scAAV.IL-1Ra administration is reasonably safe and capable of sustained therapeutic IL-1Ra production intra-articularly in joints of human scale. This profile supports consideration for human testing in OA.

Toward the treatment of osteoarthritis (OA), the authors have been investigating self-complementary adeno-associated virus (scAAV) for intra-articular delivery of therapeutic gene products. As OA frequently affects weight-bearing joints, pharmacokinetic studies of scAAV gene delivery were performed in the joints of the equine forelimb to identify parameters relevant to clinical translation in humans. Using interleukin-1 receptor antagonist (IL-1Ra) as a secreted therapeutic reporter, scAAV vector plasmids containing codon-optimized cDNA for equine IL-1Ra (eqIL-1Ra) were generated, which produced eqIL-1Ra at levels 30- to 50-fold higher than the native sequence. The most efficient cDNA was packaged in AAV2.5 capsid, and following characterization in vitro, the virus was injected into the carpal and metacarpophalangeal joints of horses over a 100-fold dose range. A putative ceiling dose of 5 × 1012 viral genomes was identified that elevated the steady-state eqIL-1Ra in the synovial fluids of injected joints by >40-fold over endogenous levels and was sustained for at least 6 months. No adverse effects were seen, and eqIL-1Ra in serum and urine remained at background levels throughout. Using the 5 × 1012 viral genome dose of scAAV, and green fluorescent protein as a cytologic marker, the local and systemic distribution of vector and transduced cells following intra-articular injection scAAV.GFP were compared in healthy equine joints and in those with late-stage, naturally occurring OA. In both cases, 99.7% of the vector remained within the injected joint. Strikingly, the pathologies characteristic of OA (synovitis, osteophyte formation, and cartilage erosion) were associated with a substantial increase in transgenic expression relative to tissues in healthy joints. This was most notable in regions of articular cartilage with visible damage, where foci of brilliantly fluorescent chondrocytes were observed. Overall, these data suggest that AAV-mediated gene transfer can provide relatively safe, sustained protein drug delivery to joints of human proportions.

OBJECTIVETo determine the outcome after early repeat celiotomy in horses operated for jejunal strangulation.STUDY DESIGNRetrospective case series.ANIMALSHorses (n = 22) that underwent repeat celiotomy for postoperative reflux (POR) and/or postoperative colic (POC) that did not improve within 48 hours from onset after initial surgical treatment of strangulating jejunal lesions by jejunojejunostomy (n = 14) or no resection (n = 8).METHODSMedical records were reviewed for clinical signs, duration of signs before repeat surgery, surgical findings and treatment, and outcome. Survival was documented by phone call at long-term follow-up. The influence of POC and POR on timing of surgery were analyzed. Long-term survival was examined by Kaplan-Meier analyses.RESULTSRepeat celiotomy was performed at a median of 57 hours after initial surgery and 16.5 hours from onset of signs, and earlier in horses with POC compared with POR (P < .05). A total of 3/22 horses were euthanatized under anesthesia. A total of 9 of 11 horses with initial jejunojejunostomy required resection of the original anastomosis due to anastomotic complications. In 8 horses without resection, second surgery included resection (4) or decompression (4). Repeat celiotomy was successful in 13/16 horses with POR. Repeat celiotomy eliminated POC in all horses (n = 9). A total of 19 horses were recovered from anesthesia and all survived to discharge. Incisional infections were diagnosed in 13/17 horses where both surgeries were performed through the same ventral median approach, and hernias developed in 4/13 infected incisions. Median survival time was 90 months.CONCLUSIONRepeat celiotomy can eliminate signs of POR and/or POC, and the additional surgery does not appear to aggravate POR. Criteria for repeat celiotomy in this study could provide guidelines for managing POC and POR after surgery for jejunal strangulation.

Magnetic resonance imaging (MRI) is the most sensitive imaging modality to detect the early changes of osteoarthritis. Currently, there is no quantifiable method to tract these pathological changes over time in the horse. The objective of this experimental study was to characterize the progression of MRI changes in an equine model of post-traumatic osteoarthritis using a semiquantitative scoring system for whole-organ evaluation of the middle carpal joint. On day 0, an osteochondral fragment was created in one middle carpal joint (OCI) and the contralateral joint (CON) was sham-operated in 10 horses. On day 14, study horses resumed exercise on a high-speed treadmill until the completion of the study (day 98). High-field MRI examinations were performed on days 0 (preosteochondral fragmentation), 14, and 98 and scored by three blinded observers using consensus agreement. Images were scored based on 15 independent articular features, and scores were compared between and within-groups. On days 14 and 98, OCI joints had significantly (P ≤ 0.05) higher whole-organ median scores (29.0 and 31.5, respectively), compared to CON joints (21.5 and 20.0, respectively). On day 14, OCI joints showed significant increases in high-signal bone lesion scores, and osteochondral fragment number and size. On day 98, high-signal bone lesion, low-signal bone lesion, osteophyte formation, cartilage signal abnormality, subchondral bone irregularity, joint effusion, and synovial thickening scores were significantly increased in OCI joints. Study results suggest that the MRI whole-organ scoring system reported here may be used to identify onset and progression of pathological changes following osteochondral injury.