Hyaluronic acid synthase-2 gene transfer into the joints of Beagles by use of recombinant adeno-associated viral vectors

Abstract

OBJECTIVE To evaluate gene transfer of recombinant adeno-associated viral (rAAV) vectors with AAV2 or AAV5 capsid and encoding hyaluronic acid (HA) synthase-2 (HAS2) into joints of healthy dogs. ANIMALS 22 purpose-bred Beagles. PROCEDURES Plasmid expression cassettes encoding canine HAS2 (cHAS2) were assessed in vitro for concentration and molecular size of secreted HA. Thereafter, rAAV2-cHAS2 vectors at 3 concentrations and rAAV5-cHAS2 vectors at 1 concentration were each administered intra-articularly into the left stifle joint of 5 dogs; 2 dogs received PBS solution instead. Synovial fluid HA concentration and serum and synovial fluid titers of neutralizing antibodies against AAV capsids were measured at various points. Dogs were euthanized 28 days after treatment, and cartilage and synovium samples were collected for vector DNA and mRNA quantification and histologic examination. RESULTS Cell transfection with plasmids encoding cHAS2 resulted in an increase in production and secretion of HA in vitro. In vivo, the rAAV5-cHAS2 vector yielded uniform genome transfer and cHAS2 expression in collected synovium and cartilage samples. In contrast, rAAV2-cHAS2 vectors were detected inconsistently in synovium and cartilage samples and failed to produce clear dose-related responses. Histologic examination revealed minimal synovial inflammation in joints injected with rAAV vectors. Neutralizing antibodies against AAV capsids were detected in serum and synovial fluid samples from all vector-treated dogs. CONCLUSIONS AND CLINICAL RELEVANCE rAAV5-mediated transfer of the gene for cHAS2 into healthy joints of dogs by intra-articular injection appeared safe and resulted in vector-derived cHAS2 production by synoviocytes and chondrocytes. Whether this treatment may increase HA production by synoviocytes and chondrocytes in osteoarthritic joints remains to be determined.

Serotype-specific transduction of canine joint tissue explants and cultured monolayers by self-complementary adeno-associated viral vectors

“…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.…”

“…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 sti e 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).…”

“…That study has evaluated the effect of conventional AAV2 and 5 encoding hyaluronic acid synthase-2 gene in canine sti e 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.…”

“…Gene transduction using conventional AAV vector has been applied in dogs for myotubular myopathy (17)(18)(19), hemophilia B (20)(21)(22), glycogen storage disease type Ia (23), retinal degeneration (24)(25)(26)(27), and mucopolysaccharidosis VII (28). For targeting joints, only a single in vivo study is available to this date regarding canine intra-articular therapy research using AAV vectors (29), while non-viral naked DNA plasmid injection (30) and an ex vivo retroviral vector transduction (31) have been utilized.…”

Exploring the Potential Feasibility of Intra-Articular Adeno-Associated Virus-Mediated Gene Therapy for Hemophilia Arthropathy

“…Given that the efficacy and safety of AAV5 for arthritis has been investigated in mouse, 59,60 rat, 61 horse, 62 monkey, 63 and proposed clinical trials (www.clinicaltrials.gov #NCT 02727764 and 03445715), it is reasonable to consider AAV5 for hemophilia. Either synovium (fibroblastlike synoviocytes) 62 or chondrocytes 64 or both 65 can be transduced by AAV5 with different tropisms. 9 In summary, direct supplementation of FVIII by IA delivery in the joint space provided protection against bleeding-induced joint damage, even in the presence of inhibitor antibodies.…”