IL-1ra gene therapy in equine osteoarthritis improves physiological, anatomical, and biological outcomes of joint degeneration

Laurie R. Goodrich Orthopaedic Research Center, C. Wayne McIlwraith Translational Medicine Institute, College of Veterinary Medicine, Colorado State University, Fort Collins, CO

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C. Wayne McIlwraith Orthopaedic Research Center, C. Wayne McIlwraith Translational Medicine Institute, College of Veterinary Medicine, Colorado State University, Fort Collins, CO

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Josh Grieger UNC Gene Therapy Center, University of North Carolina, Chapel Hill, NC

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Virginia Byers Kraus Duke Molecular Physiology Institute, Duke University School of Medicine, Durham, NC
Division of Rheumatology, Duke University School of Medicine, Durham, NC

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Thomas Stabler Division of Rheumatology, Duke University School of Medicine, Durham, NC

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Natasha Werpy Equine Diagnostic Imaging Inc, Archer, FL

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Jennifer Phillips Orthopaedic Research Center, C. Wayne McIlwraith Translational Medicine Institute, College of Veterinary Medicine, Colorado State University, Fort Collins, CO

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R. Jude Samulski UNC Gene Therapy Center, University of North Carolina, Chapel Hill, NC

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David Frisbie Orthopaedic Research Center, C. Wayne McIlwraith Translational Medicine Institute, College of Veterinary Medicine, Colorado State University, Fort Collins, CO

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Abstract

OBJECTIVE

To evaluate the effects of a gene transfer approach to IL-1β inhibition in an equine osteochondral chip fragment model of joint injury using a self-complementary adeno-associated virus with interleukin receptor antagonist transgene cassette (scAAVIL-1ra), as posttraumatic osteoarthritis in horses, similar to people, is a significant clinical problem.

ANIMALS

16 horses were utilized for the study.

METHODS

All horses had an osteochondral chip fragment induced arthroscopically in one middle carpal joint while the contralateral joint was sham operated. Eight horses received either scAAVIL-1ra or saline in the osteoarthritis joint. Horses were evaluated over 70 days clinically (lameness, imaging, and biomarker analysis) and euthanized at 70 days and evaluated grossly, with imaging and histopathology.

RESULTS

The following findings were statistically significant. Injection of scAAVIL-1ra resulted in high synovial fluid levels of IL-1ra (0.5 to 9 μg/mL) throughout the duration of the experiment (70 days). Over the duration, we observed scAAVIL-1ra to improve lameness (lameness score relative improvement of 1.2 on a scale of 0 to 5), cause suppression of prostaglandin E2 (a relative decline of 30 pg/mL), and result in histological improvement in articular cartilage (decreased chondrocyte loss and chondrone formation) and subchondral bone (less osteochondral splitting and osteochondral lesions). Within the synovial membrane of scAAVIL-1ra–treated joints, we also observed perivascular infiltration with CD3-positive WBCs, suggesting lymphocytic T-cell perivascular infiltration commonly observed with viral transduction.

CLINICAL RELEVANCE

These data provide support for further evaluation and optimization of scAAVIL-1ra gene therapy to treat equine osteoarthritis.

Abstract

OBJECTIVE

To evaluate the effects of a gene transfer approach to IL-1β inhibition in an equine osteochondral chip fragment model of joint injury using a self-complementary adeno-associated virus with interleukin receptor antagonist transgene cassette (scAAVIL-1ra), as posttraumatic osteoarthritis in horses, similar to people, is a significant clinical problem.

ANIMALS

16 horses were utilized for the study.

METHODS

All horses had an osteochondral chip fragment induced arthroscopically in one middle carpal joint while the contralateral joint was sham operated. Eight horses received either scAAVIL-1ra or saline in the osteoarthritis joint. Horses were evaluated over 70 days clinically (lameness, imaging, and biomarker analysis) and euthanized at 70 days and evaluated grossly, with imaging and histopathology.

RESULTS

The following findings were statistically significant. Injection of scAAVIL-1ra resulted in high synovial fluid levels of IL-1ra (0.5 to 9 μg/mL) throughout the duration of the experiment (70 days). Over the duration, we observed scAAVIL-1ra to improve lameness (lameness score relative improvement of 1.2 on a scale of 0 to 5), cause suppression of prostaglandin E2 (a relative decline of 30 pg/mL), and result in histological improvement in articular cartilage (decreased chondrocyte loss and chondrone formation) and subchondral bone (less osteochondral splitting and osteochondral lesions). Within the synovial membrane of scAAVIL-1ra–treated joints, we also observed perivascular infiltration with CD3-positive WBCs, suggesting lymphocytic T-cell perivascular infiltration commonly observed with viral transduction.

CLINICAL RELEVANCE

These data provide support for further evaluation and optimization of scAAVIL-1ra gene therapy to treat equine osteoarthritis.

Posttraumatic osteoarthritis (PTOA) is the most common type of arthritis in horses and dogs.1 Because PTOA is the most common cause of retirement in sport horses, it is one of the most frequently studied musculoskeletal entities in the equine species.2

Various catabolic factors have been identified as initiating PTOA, most importantly IL-1β.3 Activation of the IL-1β pathway has been correlated with cartilage catabolism and suppression of cartilage anabolism through its upregulation of matrix metalloproteinases (MMPs) including MMP-1, MMP-3, and MMP-13 and A disintegrin-like and metalloproteinases with thrombospondin motifs (ADAMTS 4 and 5), prostaglandin E2 (PGE2), nitric oxide, and other cytokines.46 These inflammation-associated factors result in the loss of glycosaminoglycans (GAGs) and collagen type II from cartilage ultimately leading to cartilage degeneration. The effects of IL-1β are highly conserved across species; induction of catabolic proteins by IL-1β has been documented in many species, particularly in human and equine chondrocytes.3,7 In people with anterior cruciate ligament ruptures, IL-1β levels are elevated and shown to be highly correlated with the severity of chondral damage from the time of anterior cruciate ligament rupture to up to 12 years following injury.8

Given that IL-1β is one of the most important inflammatory mediators in PTOA, it is logical to attempt to treat and prevent the progression of osteoarthritis (OA) with the natural antagonist to IL-1β, namely IL-1 receptor antagonist (IL-1ra). A clinical trial by Chevalier et al9 evaluated intra-articular recombinant IL-1ra protein (anakinra) for patients with OA; a single injection (150 mg) resulted in significant improvement on day 4 postinjection but no detectable improvements by weeks 4, 8, and 12 compared to 50 mg of anakinra or placebo. The authors of this report concluded that perhaps the short half-life of IL-1ra was responsible for the short duration of improvements in patients with OA.

To address the short half-life of IL-1ra and thereby potentially extend the duration of efficacy in equine patients with OA, gene delivery provides an attractive approach to increase IL-1ra levels by genetic modification of both synoviocytes and chondrocytes in situ through direct injection. Many preclinical gene therapy studies1013 have utilized various viral vectors to deliver IL-1ra to joints by both in vitro and in vivo delivery mechanisms. In the earlier preclinical gene therapy studies, viral vectors, such as retroviral and adenoviral vectors, were used; however, issues with short-term expression and local inflammation (adenoviral) as well as suboptimal transduction of tissues made these vectors less attractive when seeking long-term protein production in an often long-lasting and progressive disease.10,11 Despite these concerns, such delivery of the IL-1ra gene still resulted in a significant reduction of joint inflammation and improvement in pain and cartilage preservation in preclinical and clinical studies.12,14

In the last decade, safe and long-term expressing scAAV vectors have emerged that would be appropriate for in vivo delivery into joints. The ability to transduce synoviocytes and chondrocytes with high efficiency and produce impressive levels of therapeutic proteins both in vitro and in vivo warrant further analysis in appropriate clinical models.13,1517 In a scAAVIL-1ra equine dosing trial, therapeutic levels (10 to 1,000 times the IL-1β levels found in OA joints) of IL-1ra were measured 273 days following intrasynovial injection in healthy joints with minimal to no inflammation observed. Furthermore, in this same study, intrasynovial examination using an arthroscope that detected green fluorescent protein (GFP) revealed that scAAVGFP efficiently transduced both synoviocytes and chondrocytes within the first 30 days of injection and maintained GFP production in chondrocytes for several months.13 This is in contrast to most adenoviral gene therapeutic studies,12,18 which have only produced therapeutic levels of protein for 1 month or less within the joint.

With the historical evidence of the beneficial effects on osteoarthritic joints from delivering the IL-1ra gene in preclinical and clinical studies, we chose to use the scAAVIL-1ra vector to determine if this therapy could reduce symptoms associated with OA, with the long-term objective of testing it in phase I clinical studies in horses within the next 2 years. To study the effects (clinical, biochemical, gross, histological, and imaging outcomes) of this gene therapeutic regimen compared with a saline control, we induced PTOA using a frequently utilized equine model involving the creation of a carpal osteochondral (OC) chip fragment and subsequent strenuous exercise. Our hypothesis was that scAAVIL-1ra gene therapy could result in significantly enhanced intrasynovial production of IL-1ra in joints in which OA was induced and high levels of therapeutic protein would occur for the duration of the study. Further, we hypothesized that scAAVIL-1ra gene therapy would result in improvement in clinical outcomes such as pain (reflected by lameness), intrasynovial biochemical markers such as PGE2, gross scores of cartilage and synovial membrane, histological scores of hyaline cartilage and subchondral bone, and radiographic and MRI features of OA and demonstrate critical endpoints that would support initiation of clinical studies.

Methods

Experimental design and induction of OA

Sixteen skeletally mature horses, geldings and females, of mixed breed, aged 2 to 5 years with no radiographic abnormalities or signs of lameness were approved by the IACUC (No. 10-1613A) for use in this study. Horses were between 400 and 450 kg. Each horse underwent bilateral arthroscopic surgery of the middle carpal joints (Figure 1) following anesthesia.2 One randomly selected middle carpal joint acted as a control, and an 8-mm OC fragment was created in the contralateral limb. To create debris in the joint and to ensure fragments did not immediately heal to the parent bone, an approximately 15-mm-wide defect bed was made between the fragment and parent bone using a motorized arthroburr12 without ingress or egress of fluid. The contralateral joints were sham operated, and joint surfaces were examined to ensure the absence of joint injury. Horses were box stall rested and bandaged until suture removal at 10 days postsurgery.

Figure 1
Figure 1

Equine middle carpal joint illustration, experimental design, and study timeline. An illustration (A) of the equine middle carpal joint indicating the area where the osteochondral chip fragment was induced (close-up view shown to the left). The boxes indicate areas in which samples were taken for glycosaminoglycan (GAG) analysis (CI) and cartilage histology (CR, CU, C3, and C4). Solid, vertical, rectangular boxes indicate areas in which osteochondral blocks were harvested for histological assessment of cartilage and subchondral bone. Diagram modified from Frisbie et al, Gene Therapy, 2002 with permission. A diagram illustrating the experimental design (B) indicates the absence or presence of an osteochondral chip fragment, and the administration of saline or scAAVIL-1ra treatment 14 days following surgery. The study timeline (C) illustrates time points at which treatments were administered, and data and clinical samples were collected. Flags above the timeline indicate points in which lameness exams were performed, and blood and synovial fluid aspirates were collected. Flags below the timeline indicate points in which radiographs and MRIs were performed. OA = Osteoarthritis.

Citation: Journal of the American Veterinary Medical Association 262, S1; 10.2460/javma.24.02.0078

Treatment groups

Ten days following fragment creation, lameness exams were performed by a single examiner (same examiner throughout the study) unaware of the treatment group and horses were stratified so that grades of lameness were apportioned equally among the treatment and control groups. Horses in the control group were given 5 mL of 0.9% saline intra-articularly in both middle carpal joints (OA-affected and non-OA joints) on day 14. Horses in the treatment group received serotype 2 scAAVIL-1ra (n = 8; 5 X 1012 viral particles) diluted in 5 mL of saline in the OA-affected middle carpal joint and 5 mL of saline IA in the non-OA middle carpal joint (8; control group). The adeno-associated virus used for this study (bioreactor propagated scAAV) has been evaluated for optimal dosing conditions and has been previously described.13

Exercise

Horses were housed in stalls (3.65 m X 3.65 m) and began a strenuous exercise regimen on a high-speed treadmill at 14 days postsurgery. Each horse was trotted for 2 minutes (16 to 19 km/h), galloped for 2 minutes (32 km/h), and trotted for 2 minutes (16 to 19 km/h), 5 days per week until day 70.

Clinical outcomes

Clinical examinations of both forelimbs were made by a specialist unaware of the treatment group with expertise in clinical evaluation at day 0 and weekly after day 7 until the end of the study period. Lameness (pain) was graded on a standardized scale of 0 to 5,19 and other parameters (synovial effusion and joint flexion) were graded on a scale of 0 to 3 (0, normal; 3, severe). Flexion was the amount (ordinal scale) of lameness worsening after flexing the joint for 30 seconds. Range of motion was also measured using the goniometer placed on the limb in the same place for every horse with permanent marking (nail polish). The limb was flexed, and the degree was read on the device. Joint circumference was also measured with a tape measure at the level of the middle carpal joint.

Synovial fluid and serum

Synovial fluid was collected at the time of surgery (day 0), and weekly after day 14 until the termination of the study (day 70). An aliquot of synovial fluid was assessed immediately for color, clarity, total protein, and fluid cytology using routine clinicopathologic parameters. Remaining synovial fluid aliquots were stored at –80 °C until the end of the study when they were analyzed for the presence of IL-1ra (Biotechne), PGE2 (Enzo Scientific), GAG (Sigma), bilirubin/biliverdin, N-terminal telopeptides (NTx; IDDS), and C-terminal telopeptides of collagen type II (CTX-II; Cloud-Clone). Serum was collected on all horses and assessed for the presence of IL-1ra and GAG.

Gross pathological observations of joints

Horses were humanely euthanized with intravenous sodium pentobarbital. Forelimbs were removed mid radius for MRI analysis and were aseptically prepped for gross examination. Middle carpal joints were disarticulated and subjectively scored by a single observer unaware of the treatment group. Scores were on a scale of full and partial thickness cartilage erosion, hemorrhage, and synovial adhesions as previously described.20 Articular cartilage samples were collected from areas of the joint depicted (Figure 1). Osteochondral sections were taken from the radial and third carpal bones for H&E and safranin-O/fast green staining. Synovial membrane samples were taken from the area closest to the defect and were assessed with H&E staining.

Histological examinations

Synovial membrane, articular cartilage, and OC samples were placed in a 10% neutral buffered formalin solution for 48 hours. Osteochondral samples were decalcified in Formical solution (American MasterTech) and monitored daily until decalcification was complete based on the pliability of samples. All samples were processed, embedded, and mounted onto charged slides. Sections were serially hydrated in ethanol followed by staining with either Harris hematoxylin for 10 minutes and eosin Y counterstain, or Weigert’s hematoxylin and 0.1% aqueous safranin-O for 7 minutes and 0.2% aqueous fast green counterstain. Equine trachea was used as a staining control.

The H&E-stained synovial membrane sections were blindly evaluated for cellular infiltration, intimal hyperplasia, subintimal edema, subintimal fibrosis, and vascularity as previously described.20 The H&E-stained articular cartilage sections were evaluated for chondrocyte necrosis, chondrone formation, fibrillation, and focal cell loss. All assessments for cartilage and subchondral bone were made as previously described.20 Silver staining was performed to determine the presence of lymphocytes within the synovium.

Articular cartilage matrix evaluation

Total articular cartilage GAG content of the radial and intermediate carpal samples was assessed using the 1,9-dimethyl-methylene blue technique as described by Farndale et al.21 Total GAG was reported as micrograms per milligrams of tissue dry weight. Cartilage matrix metabolism was also assessed in the intermediate carpal samples with the use of a radiolabel as previously described22 and reported as counts per minute per milligrams of tissue dry weight.

Diagnostic imaging

Radiographic evaluations (dorsal-palmar and flexed lateral views) were performed on days 0, 14, and 70 to assess changes in the joint capsule attachments, as well as in the subchondral bone, and periarticular osteophyte formation according to the previously described ordinal scale.23 In brief, images were evaluated on a scale of 0 to 4 (0 = normal; 4 = severe change) categorically for bony proliferation at the joint capsule attachment, subchondral bone lysis, and osteophyte formation. The MRI evaluations were performed on days 5 and 70 to assess synovial fluid and synovial proliferation, joint capsule thickness, edema and fibrosis, and edema and sclerosis of the radial and third carpal bones. The MRIs (OrthOne 1.0 T MRI; ONI GE) were performed by a board-certified radiologist unaware of treatment groups and parameters scored as previously described.24

Statistical analysis

Mixed model ANOVA (SAS, Version 9.2; SAS Institute Inc) was used to analyze the results. Data transformation was performed and reported if residual plot construction did not meet the fulfillment of model assumptions. Least squares mean procedure was utilized (SAS, Version 9.2) for individual comparisons. Except where otherwise specified, a P value less than .05 was considered significant. Dependent variables measured at multiple time points were analyzed using the following independent variables: sample collection day, presence or absence of a fragment within the joint/limb, and treatment (scAAVIL-1ra or saline). The interaction between the chip and horse within treatment variables was used as a random effect variable. Day of collection was also used in the repeated statement with interaction between and within treatment variables acting as the subject, using type III autoregressive covariance matrix. This was also utilized for dependent variables measured at 2 or fewer time points; however, the model statement only utilized the presence of chip, treatment, and interaction between these variables, whereas the random statement only used the subject within treatment variables.

Results

In this equine translational model of PTOA, the middle carpal joint in every horse had a fragment created and the contralateral joint was sham operated and served as a control, non-OA joint (Figure 1). Therefore, there were 8 horses that received intra-articular scAAVIL-1ra into the joint in which PTOA was induced and 8 control horses that received 5 mL of saline into the joint in which PTOA was induced. The 70-day timeline and outcomes measured are shown. The successful induction of OA was confirmed in this model based on consistent clinical outcomes, synovial fluid analyses, postmortem examinations, and histologic evaluation of synovial membrane, cartilage, and subchondral bone. When treatment is reported below, it refers to serotype 2 scAAVIL-1ra–treated PTOA joints compared to untreated (saline injected) PTOA joints.

Clinical outcomes

Clinical outcomes were consistent with the induction of PTOA for all joints in which it was induced. Based on outcome assessments starting 14 days after PTOA induction, there were significant increases in lameness (P < .001), lameness following joint flexion (P < .007), effusion (P < .001), and joint circumference (P < .001) and a decrease in degrees of flexion measured with a goniometer (P < .001). Treatment of OA limbs with scAAVIL-1ra resulted in significant (P = .034) improvements in lameness scores compared to saline-treated OA limbs over the duration of the study with the greatest improvement in lameness (from the time of injection of either scAAVIL-1ra or saline) by day 70 (Figure 2). Treatment did not result in significant improvements in joint circumference or effusion scores.

Figure 2
Figure 2

Improvement in lameness over time. The osteoarthritis (OA) joints of the treated horses (right) showed significant improvement compared to those of the untreated horses (left) over a 10-week study period. Middle carpal joints of untreated horses (both OA and sham-operated received saline) and middle carpal joints of scAAVIL-1ra–treated horses received treatment and sham-operated joints in this group received saline. Box plots demonstrate median (horizontal lines), and upper and lower ranges are illustrated by upper and lower extents.

Citation: Journal of the American Veterinary Medical Association 262, S1; 10.2460/javma.24.02.0078

Synovial fluid and serum biomarkers

Results for biochemical markers were also consistent with PTOA induction in this model. In both scAAVIL-1ra– and saline-treated joints, mean synovial fluid concentrations increased significantly in CTXII and NTX (reflecting degradation of types II and I cartilage from articular cartilage), biliverdin/bilirubin (consistent with hemarthrosis), and total protein (reflecting a joint exudate that is a sign of a local change of the capillary permeability in the joint capsule).

Treatment with intra-articular scAAVIL-1ra day 14 after PTOA induction resulted in significant elevations (P < .0001) of IL-1ra protein within 7 days following the intra-articular injection (Figure 3). The SEs at each time point are provided (Supplementary Figure S1). From induction of disease until completion of the study, the mean IL-1ra concentration within the treated joints remained 1,009-fold higher than the mean concentration in the untreated joints (4.6 X 106 ± 2.9 X 106 SE pg/mL in the treated joints vs 4.7 X 103 ± 1.2 X 106 SE pg/mL in the untreated joints). No significant changes of IL-1ra in the serum were detected.

Figure 3
Figure 3

The IL-1 receptor agonist (IL-1ra) protein concentration in synovial fluid over time. The IL-1ra concentrations are reported as log of picograms per milliliter. Following injection on day 14, levels remained significantly increased throughout the study (an average of 1,009-fold compared to the osteoarthritis [OA] untreated joints). A figure with SE is provided (Supplementary Figure S1).

Citation: Journal of the American Veterinary Medical Association 262, S1; 10.2460/javma.24.02.0078

In addition to elevations in IL-1ra levels in treated joints, significant (P < .0006) declines in mean PGE2 levels were detected over time (Supplementary Table S1). Further, intra-articular total protein was also significantly decreased (P = .007) most specifically on day 63 following PTOA induction (Supplementary Table S2).

Postmortem examination: gross pathology

Confirming that induction of PTOA was successful in this model, all joints in which an OC fragment was created resulted in some level of pathologic change compared to the sham-operated control (Figure 4). This was evidenced by partial or full-thickness erosions at both the site of OC fragment creation and remote from the area where the OC fragment was made. ScAAVIL-1ra–treated joints had improved partial, full, and total erosion scores compared to untreated joints; however, differences between the treated and nontreated joints were not significant.

Figure 4
Figure 4
Figure 4
Figure 4

Full thickness, partial thickness, and total cartilage erosion scores. Osteoarthritis (OA) joints had significantly higher (worse) scores than sham-operated joints for full thickness (A), partial thickness (B), and total erosion scores (C); however, there were no significant differences between untreated and treated horses. a–cAsterisks depict significant differences, superscripts depict similarities or significant differences between groups.

Citation: Journal of the American Veterinary Medical Association 262, S1; 10.2460/javma.24.02.0078

Postmortem examination: histologic evaluation of cartilage

The induction of PTOA in this model was successful (as assessed from the weight-bearing surfaces of the radial, third and fourth carpal bone) and caused significant (P < .05) cartilage fibrillation, chondrone formation, chondrocyte necrosis, and focal cell loss on the radiocarpal bone and third carpal bone. Further, based on safranin-O/fast green staining, GAG content was decreased (worsened) with PTOA. Based on summation scores of cartilage from the radiocarpal bones, treatment with scAAVIL-1ra was associated with significantly better histological outcomes compared to PTOA joints that were untreated (Figure 5). The greatest treatment effects were observed for chondrone formation and focal cell loss subscores.

Figure 5
Figure 5
Figure 5
Figure 5

Chondrone formation, focal cell loss, and summation scores in radial carpal cartilage samples. Summation scores in osteoarthritis (OA) and sham-operated joints reveal that OA joints in scAAVIL-1ra–treated joints were significantly lower (improved) than OA-untreated joints (A). Images of chondrone formation in cartilage from OA-untreated (Ba and Bb) and -treated (Bc and Bd) radial carpal samples, as well as focal cell loss in cartilage from OA-untreated (Ca and Cb) and -treated (Cc and Cd) radial carpal samples. Boxed areas (Ba and Bc; Ca and Cc) are shown at higher magnifications (Bb and Bd; Cb and Cd). Arrows indicate an increase in the formation of chondrones (Bb) and focal cell loss (Cb) in the untreated samples. There was a significant decrease (improvement) in the summation scores of OA-treated cartilage samples (A). Asterisks depict significant differences, superscripts depict similarities or significant differences between groups. Summation scores are a compilation of fibrillation, chondrone formation, chondrocyte necrosis, and focal cell loss; chondrone formation and focal cell loss contributed the greatest change (worsening) in these scores (B and C).

Citation: Journal of the American Veterinary Medical Association 262, S1; 10.2460/javma.24.02.0078

Postmortem examination: histologic evaluation of subchondral bone

As with cartilage scores, creation of PTOA in this model was consistent with subchondral bone pathology in OC-fragmented joints. Subchondral bone scores were significantly (P < .05) worse as defined by worse cartilage attachment, OC lesions, subchondral bone remodeling, and OC splitting. Treatment of OA joints with scAAVIL-1ra resulted in significant (P < .034) improvements in subchondral bone scores compared to untreated joints (Figure 6). Specifically, treatment reduced the formation of OC lesions and OC splitting.

Figure 6
Figure 6
Figure 6
Figure 6

Osteochondral lesions, osteochondral splitting, and summation scores in osteochondral samples. Summation scores in osteoarthritis (OA) and sham-operated joints reveal that OA joints in scAAVIL-1ra–treated joints were significantly lower (improved) than OA-untreated joints (A). Osteochondral lesions were evaluated in OA-untreated (Ba and Bb) and OA-treated (Bc and Bd) samples, and osteochondral splitting was evaluated in OA-untreated (Ca and Cb) and OA-treated Cc and Cd) samples. Boxed areas (Ba and Bc; Ca and Cc) are shown at higher magnifications (Bb and Bd; Cb and Cd). Arrows indicate an increase in the presence of osteochondral lesions (Bb) and osteochondral splitting (Cb) in OA-untreated joints. Asterisks depict significant differences, superscripts depict similarities or significant differences between groups. Summation scores are a compilation of cartilage attachment, osteochondral lesions, subchondral bone remodeling, and osteochondral splitting; osteochondral lesions and osteochondral splitting contributed to the greatest change (worsening) in these scores.

Citation: Journal of the American Veterinary Medical Association 262, S1; 10.2460/javma.24.02.0078

Postmortem examination: histologic evaluation of synovial membrane

Consistent with induction of PTOA with OC fragmentation, synovial summation scores (cellular infiltration, intimal hyperplasia, subintimal edema, subintimal fibrosis, and vascularity), were significantly worse (P < .001) in OA joints compared with sham-operated control joints. In contrast to cartilage and subchondral bone summation scores, summation scores of the synovial membrane of OA joints treated with scAAVIL-1ra resulted in higher (worse) scores compared to PTOA joints that were untreated (P < .021) (Supplementary Figure S2). This was due mainly to perivascular cellular infiltration (perivascular cuffing) and also synovial fibrosis.

Immunohistological staining (silver staining) performed on the histological samples where perivascular cuffing was identified revealed mostly T-cell infiltration (Supplementary Figure S3) without the increased thickness of the synovial lining.

Imaging: MRI assessment

The MRI confirmed the establishment of PTOA in OC-fragmented joints based on significant (P < .001) increases in joint capsule edema, fibrosis and thickening, and radiocarpal bone sclerosis. ScAAVIL-1ra did not result in any significant improvements in MRI scores in OA-treated joints compared to untreated OA joints.

Imaging: radiographic assessment

Radiographic examinations performed on day 0 (before OC fragment creation), day 14, and day 70 revealed the expected worsening of scores with the creation of the OC fragment confirming successful PTOA induction and progression associated with this model. Radiographic features noted in this model system included increased subchondral bone sclerosis and osteophyte formation compared to the sham-operated joints. Consistent with the worsening of flexion scores seen on physical outcome parameters and the histopathological changes in the synovial membrane, treatment of OA joints with scAAVIL-1ra resulted in significantly (P < .0042) worse scores for enthesophyte formation evaluated radiographically (change from baseline of 1.9 ± .76 for treated vs 0.5 ± 0.1 untreated on a scale of 0 to 4).

Discussion

In this well-established equine model of PTOA, we achieved long-term production of therapeutic levels of IL-1ra in the PTOA joints injected with AAVIL-1ra. These results are consistent with the majority of AAV in vivo studies in other organ systems.25 Important and positive effects of treatment on PTOA joints included significant improvements in lameness, decreased PGE2 concentrations in synovial fluid (related to improvements in lameness and inflammation as described below), decreased cartilage degeneration (specifically a reduction in chondrocyte loss and chondrone formation), and significant improvements in subchondral bone histology (OC splitting and lesions). As observed in prior gene therapeutic studies12,18 using adenovirus, angiocentric lymphocytic infiltration, termed perivascular cuffing, occurred around the synovial vessels. Interestingly, this finding was not accompanied by synovial membrane thickening but by the occurrence of some fibrosis within the subsynovium. Based on immunostaining, cells were specifically “T” lymphocytes commonly associated with viral infections. It is noteworthy that robust IL-1ra protein production was present associated with this finding suggesting that the T lymphocytes did not damage the cells that were producing IL-1ra. Interestingly, perivascular cuffing has not been observed when the same dose of viral vector (5 X 1012 viral particles) was administered to normal joints of horses with the same vector and transgene.13

The synovial fluid IL-1ra levels in PTOA joints in this study were much higher than levels achieved in normal (noninflamed) joints injected with the same dose of viral vector with the vector backbone and transgene unchanged from this study.13 We presume this was a result of upregulation of the cytomegalovirus (CMV) promoter, used in this vector construct, in the context of inflammatory mediators within the joint.26,27 Two different studies in horses27 and rats28 described elevated levels of GFP detection in OA joints versus non-OA joints; however; these observations were qualitative assessments and not quantified as in this study. Only 2 other studies have revealed elevated protein levels when the CMV promotor was upregulated with inflammation. The first was a study by Kornegay et al,29 where the CMV promoter upregulated protein expression in muscle with inflammation, and the second by Watson Levings et al,30 where highly upregulated production of IL-1ra in inflamed joints relative to normal joints was also reported in an OC fragment preclinical model in horses. Importantly, these results suggest that in the future, sufficient IL-1ra could be produced in a PTOA joint from a much lower dose of viral vector. This might have the added benefit of minimizing the T-cell response seen in this study providing a better safety margin when treating humans. This is likely a novel and important finding to consider in optimizing a phase I clinical trial design for humans; however, additional studies will be required to confirm this hypothesis.

The clinical improvement of lameness in OC-fragmented limbs injected with scAAVIL-1ra may have been due to the reductions in PGE2 levels thereby suppressing nociceptive stimuli.31 PGE2 has historically been the inflammatory mediator in synovial fluid examined in this equine model because the assay to measure this cytokine is well-established and reliable in the horse, accurately reflects levels of inflammation, and has been correlated to clinical pain as well as identified as a marker of inflammation.12,22 The suppression of PGE2 would most likely be due to the binding of IL-1ra to the IL-1 receptors, thus inhibiting the catabolic cascade initiated by IL-1β in inflamed joints.3,12,32 While the PGE2 was higher at day 14 in the treated OA joints than the untreated OA joints, significant declines resulted in an important change in this mediator.

While it is unknown exactly how much IL-1ra is required to block all or most of the binding of IL-1β to IL-1 receptors, levels needed to have a clinically therapeutic effect using protein delivery are estimated to be approximately 10 to 1,000 times the levels of IL-1β in the synovial cavity.33 For the 70 days following injection, the levels of IL-1ra far surpassed any other successful preclinical or clinical study using either therapeutic effects or recombinant IL-1ra.12,17,34,35 Clinical studies9 utilizing 150 mg of IL-1ra recombinant protein intra-articularly started out with high IL-1ra levels (mg/mL), but because the protein was recombinant and short lived, therapeutic effects diminished quickly. Further studies are needed to better understand the levels of IL-1ra needed to inhibit IL-1β in inflamed and osteoarthritic joints.

Histopathological findings of the articular cartilage from joints injected with scAAVIL-1ra revealed improvements based on reductions in chondrocyte loss and chondrone formation. Both of these findings are consistent with suppression of detrimental cytokine production locally within the joint.2,20,36 Further, the significant effects of scAAVIL-1ra on subchondral bone in the context of induced OA revealed that treatment was beneficial for both articular cartilage and subchondral bone. These important effects on subchondral bone have not been documented in previous in vivo terminal intra-articular gene therapeutic studies, which thoroughly evaluate bone and offer an exciting method for treating the often significant subchondral bone pathology associated with PTOA.

Recent studies37,38 reveal that cytokines, such as IL-1β, and proteases produced in cartilage alter the function of osteoblasts within subchondral bone and induce cells with an osteoblastic phenotype to switch to a sclerotic phenotype, thus resulting in subchondral bone sclerosis. By decreasing inflammation (either directly or indirectly) with scAAVIL-1ra gene therapy we appear to have induced a therapeutic effect on subchondral bone. This could have significance for all stages of OA because maintenance of subchondral bone integrity is an important component of treatment.39 To the authors’ knowledge, current intra-articular treatments such as corticosteroids, biologics, and stem cell therapy have not resulted in improvements in subchondral bone healing suggesting a niche role for IL-1ra therapy in PTOA.

Synovial membrane findings on histological examination revealed a lymphocytic “perivascular cuffing” of vessels within the synovium. This finding has been identified with animal studies that have utilized adenovirus (vs AAV) to deliver therapeutic proteins to joints.12,18 We did not expect to see the perivascular cuffing with AAV that is typically associated with adenovirus because normal (non-OA) joints in a previous study40,41 using AAV by our group did not reveal this perivascular cuffing. Possibilities for this histological finding include (1) the optimized DNA sequence constructed for the equine IL-1ra made some proinflammatory foreign protein based on an alternative open reading frame within the DNA sequence,13 or (2) the capsid in an inflamed joint provoked an immune response.42 To address the first option, we analyzed the optimized equine IL-1ra sequence, and no alternative DNA sequences could be identified that would be transcribed into a foreign mammalian protein. Although AAV and specifically scAAV can be mild immunogens,43,44 when T-cell responses have been identified in clinical trials, such as Brantly et al,45 the protein expression from the transgene was not inhibited, suggesting that the presence of T lymphocytes did not mount a destructive effect on the cells that were transduced. This suggests that although T-cell responses to the vector may exist in some cases, this response does not impact transgene expression.

This equine model of PTOA has been tested in over 20 preclinical trials intended to investigate PTOA therapies, including a trial for constitutive delivery of IL-1ra protein as well as adenoviral IL-1ra gene therapy.2,12,34,46 Interestingly, in the present study, higher levels of IL-1ra were detected for longer periods of time than that of the study of Frisbie et al12; however, clinical effects in the current study were not as potent.12 We believe this may be due to a scAAV dose that was higher than needed, which resulted in some detrimental synovial changes, borne out in the increased flexion scores, histological (perivascular cuffing), and radiographic scores (increased osteophyte scores) of scAAVIL-1ra–treated joints. The positive effects noted in lameness outcome, total protein levels, and cartilage as well as subchondral bone were encouraging despite some of the nonadvantageous outcomes.

Current studies in humans and preclinical studies in animals suggest that IL-1ra administered as (1) recombinant protein (anakinra), (2) concentrated from the patient’s own blood (autologous conditioned serum), or (3) gene therapeutic approaches, results in anti-inflammatory and chondroprotective effects.12,14,4749 It is well understood that the crux of successful IL-1ra therapy has been to stimulate high, therapeutic levels of this protein for long periods (> 4 weeks) of time.9 When this treatment has failed it has been surmised that levels of IL-ra protein simply did not stay at “therapeutic” concentrations for long enough periods due to short half-lives of the therapy when administered as a recombinant protein or via gene therapy using a vector that produces protein for short periods only (< 4 weeks).9,50 The work presented here suggests that IL-1ra delivered through AAV gene therapy resulted in net positive outcomes for PTOA. Further work to diminish any negative outcomes will be ongoing so that future clinical studies can be safely performed to determine its long-term efficacy.

In conclusion, confirming our hypothesis, scAAVIL-1ra gene therapy in this equine model of OA resulted in apparent therapeutic or supra-therapeutic levels of IL-1ra protein for the duration of the study. To our knowledge, no OA gene therapy studies have attained these levels of IL-1ra for this duration (70 days). In addition, some outcome parameters improved with treatment as evidenced by lameness improvement, PGE2 reduction in the joints, and improved cartilage and subchondral bone histology. However, vector administration resulted in lymphocyte perivascular cuffing and associated synovial fibrosis and osteophyte formation in this disease setting. Importantly, this study revealed that most likely, 10-fold lower doses of therapeutic vector could be administered in an injury or OA trial due to the likely upregulation of the CMV promoter in the context of inflammation.29 Further dosing in OA joints of large animals, specifically horses with both surgically induced and naturally occurring OA, should identify technical steps as well as vector modification (increase or decrease of dose levels) that could improve safety and allow this potentially promising treatment to be administered to patients with PTOA and OA.

Supplementary Materials

Supplementary materials are posted online at the journal website: avmajournals.avma.org.

Acknowledgments

All data associated with this study are available in the main text or the Supplementary Materials.

The authors thank the staff of the Orthopaedic Research Center at CSU for their care of the horses in this study and Skyla Hall for her assistance with editing.

Disclosures

The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.

Funding

This work was supported by NIH grant No. 1K08AR054903-01 A2 (LRG, CWM, and RJS), Grayson Jockey Club Research Foundation Award (LRG, CWM, RJS, NW, and DFF), and NIH grant No. R01 AI117408 (RJS).

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