To develop effective gene therapy for OA in dogs, ideally the optimal scAAV vector serotype in joint tissues should be determined first. We therefore designed this in vitro study before applying scAAV vectors in vivo into canine joints. To our knowledge, this is the first study to compare transduction efficiencies of scAAV vectors in canine synoviocytes, chondrocytes, MSCs, cartilage and synovial explants. MSCs were also included in this experiment since we wanted to target joint cells and MSCs for treating OA. Our hypothesis is rejected since we revealed different transduction efficiencies to canine joint cell monolayers and explants among different scAAV vector serotypes. Based on the result of the current study, the most promising scAAV serotypes are scAAV2 and 2.5.
Previous canine joint gene therapy research described the use of various techniques and includes the encoding of genes of anti-inflammatory cytokines to modify the joint disease process in vivo. One study used an indirect gene delivery technique to inject cultured synovial fibroblasts, which were transduced ex vivo by a retroviral vector encoding human interleukin-1 receptor antagonist (IL-1ra), into cranial cruciate ligament deficient canine stifles (31). The injection increased the local production of IL-1ra in the stifle joint and reduced the progression of OA (31). A more recent study used a plasmid DNA injection technique that directly delivers IL-10 gene into canine joints (30). This double-blind, prospective, randomized, placebo-controlled pilot study included 10 dogs with naturally occurring canine OA. Pain scores decreased based on subjective veterinary and owner questionnaires without any complications during the 8 weeks of the study period (30). The AAV vector system (e.g., conventional AAV and scAAV) in canine joints has also been investigated in laboratory settings and live animals. scAAV vectors package double-stranded genomes allowing faster transduction rate via bypassing the rate-limiting DNA synthesis step, while conventional AAV vectors package single-stranded genomes requiring synthesis of the complementary strand in the host cell (32, 33). Santangelo et al. revealed successful transduction of normal and osteoarthritic canine cartilage explants with scAAV2 and conventional AAV2 vectors (42). Our study is in agreement with these results, revealing good transduction efficiency with scAAV2 in dogs’ joint cell monolayers and tissue explants.
At the time of writing, our literature search reveals only one study in live dogs describing intra-articular gene therapy using conventional AAV and none using scAAV. That study has evaluated the effect of conventional AAV2 and 5 encoding hyaluronic acid synthase-2 gene in canine stifle joints without a formal screening of AAV serotypes (29). Interestingly, it revealed that conventional AAV5 had consistent gene transfer in a dose-dependent manner, while conventional AAV2 had inconsistent gene transfer with a lack of dose-response effect (29). This result is worth noting since conventional AAV2, which showed successful transduction to canine joint cells in vitro (42), did not show consistent transduction in vivo. Similar mismatched gene transduction efficiency between in vitro and in vivo studies has been reported in other species. Watson et al. compared the transduction rate of conventional AAV1, 2, 5, 8, and 9 encoding human IL-1ra in equine synovial fibroblast culture and live horses’ joints (43). While serotype 1, 2, and 5 showed superior IL-1ra production in cell culture, serotype 2, 5, and 8 showed better IL-1ra production in joints (43). There is no clear explanation for why the serotypes act differently in vitro and in vivo; however, cell surface glycan receptor expression (36) and unforeseen immunity (38) are possible reasons for these differences.
Since there is scarce information regarding behaviors of scAAV serotypes in canine joints, this in vitro scAAV serotype screening study was necessary as the first step to minimize the use of live animals for our next in vivo study due to various serotypes, even with an understanding of which those serotypes can act differently in vitro and in vivo. Consistently good transduction rates of scAAV2, 2.5 and 5 both in vitro and in vivo in horses justify the use of in vitro serotype screening experiments in dogs for selecting serotypes for in vivo experiments (37, 43).
For successful transduction of scAAV vectors, cell surface glycans are the crucial first step for the vectors to attach to the surface of target cells. It has been shown that AAV2, 3, and 6 bind to heparan sulfate as the primary receptor, AAV1, 4, 5, and 6 bind to sialic acid, AAV8 binds to laminin receptor in the basement membranes of many tissues, and AAV9 binds to glycans with terminal galactose (44–46). Cell surface receptor expression can be affected by various factors, including oxygen concentration, growth medium, culture system, or cell density (47), even by arthritic conditions (48). We classified the scAAV vectors into three classes based on different levels of transduction efficiency between cell monolayers and tissue explants as previously established from our work (36). There may be different expressions of cell surface receptors in joint cells in the native environment. Cell surface receptor analysis using flow cytometry with receptor antibodies (43) or comparison of transduction with enzymatic digestion of cell surface receptor (36) can be considered to identify the receptor expression in each condition for further investigation.
Another factor that can affect transduction efficiency in vivo is the innate and acquired immunity of live animals towards scAAV vectors. Serum neutralizing antibodies, which can make injected scAAV vectors ineffective, can exist naturally or can be formed with repeated injections. Immune response regarding scAAV vectors has not been investigated in dogs, but it may have similar response to that of conventional AAV vectors due to same capsid structure. The most prevalent neutralizing antibody in dog serum is against AAV6 (49–51). Antibodies against AAV5, 8, and 9 have not been reported in dogs (49, 51). However, lower concentrations of canine antibodies against AAV1(49, 50) and AAV2 (50), compared to those of AAV6, have been reported. In addition, a strong humoral and cellular immune response has been revealed in vivo application of conventional AAV2 into canine muscle (52). It is unknown whether the same immune response would occur intra-articularly. However, the immune response against AAV2 may be related to the inconsistent transduction rate of AAV2 in Kyostio-Moore et al.’s experiment (29). Immune response regarding AAV2.5 has not been reported to this date.
scAAV2.5 is an enhanced chimeric vector, combining characteristics of serotype 1 and 2 (53). scAAV2.5 is designed to bind to heparan sulfate to have an affinity to tissue well like scAAV2, but also have the ability to avoid immune response seen against scAAV2 via features of scAAV1 (53). Goodrich et al. revealed scAAV2.5 produced encoded protein over a more prolonged period than scAAV2 (i.e., 6 months and 23 days in scAAV2.5 and 2, respectively) in horses, while both scAAV2 and 2.5 showed effective transduction with high levels of protein concentration intra-articularly during those periods (37). Therefore, scAAV2.5 may be the more suitable vector for in vivo application than scAAV2 in dogs.
A major difference between the current research from our previous equine research was overall transduction efficiency. Previously reported transduction efficiencies of scAAV2, 3, 5 and 6 in 4 000 vpc at day 7 in equine chondrocytes and synoviocytes were more than 95% and 85%, respectively (34). Conversely, for scAAV2, 2.5, 3, 5 and 6 in 10 000 vpc at day 4 they were approximately 75%, 70%, 4%, 11%, and 20% in canine chondrocytes and 47%, 38%, 2%, 12%, and 3% in synoviocytes, respectively. Even though we are comparing canine results on day 4 and equine results on day 7 post-transduction, there still appears to be a substantial difference between the two species. Similarly, Watson et al. revealed that equine joint cell monolayers showed higher transduction efficiency than human joint cell monolayers (43). Interestingly, heparan sulfate glycan expression level was significantly higher in equine joint cells than in humans, and 20-fold or more of the viral genome was detected within equine joint cells than in humans for the same viral particles per cell (43). As there is an innate difference between equine and human cells, there may be a similar difference between canine and equine cells.
Transduction efficiencies of cell monolayers appeared to be superior to those of tissue explants from the current study. Possible reasons include different expression levels of cell surface receptors, various amounts of extracellular matrix (ECM), and the need of diffusion of scAAV vectors into tissue explants. Given the small size (~ 20 um) of the scAAV particle, the latter rationalization seems less likely. Further investigation with enzymatic digestion of cell surface receptor or ECM would allow to understand the cause of the difference between cell monolayers and tissue explants.
Another interesting finding of this study is that GFP expression was observed only from the superficial zone of cartilage explants, not the deeper zones. This could potentially be a reason why transduction efficiency of cartilage explants was substantially lower than other culture types in flow cytometry. According to Santangelo et al., while normal cartilage showed uniform GFP expression in partial or full-thickness cartilage, osteoarthritic cartilage showed GFP expression only in the superficial zone (e.g., tangential and transitional zone) of cartilage, not the deeper zones (42). Our results differ from these findings however, we used normal cartilage only. It has been suggested that chondrocytes in the superficial layers are metabolically more active to repair damaged matrix if needed (54). Chondrocytes in the deeper zone may have unique characteristics that inhibit AAV vector transduction or have a slower metabolic rate that cannot achieve transduction. Further comparison between normal and osteoarthritic cartilage is warranted on scAAV transduction.
There are several limitations of this study. First, due to the in vitro nature of the study, these results may not represent gene transfer in live dogs’ joints. This study design is helpful to screen many serotypes without using live animals, but they do not necessarily exactly reflect the native environment of joints. In this respect, the application of these vectors in vivo may provide more representative information on the transduction efficiency of variant AAV serotypes. Second, a small number of samples were used for analysis, which could potentially cause a Type I statistical error. Third, the age of the tissue donor and the type of joints were not controlled. Lastly, even though the neoplasia was not at the location we harvested tissue, the collected tissues may not represent normal healthy tissue since some were harvested from dogs had neoplasia.