Persistence, Localization, and External Control of Transgene Expression After Single Injection of Adeno-Associated Virus into Injured Joints

Abstract

A single intra-articular injection of adeno-associated virus (AAV) results in stable and controllable transgene expression in normal rat knees. Because undamaged joints are unlikely to require treatment, the study of AAV delivery in joint injury models is crucial to potential therapeutic applications. This study tests the hypotheses that persistent and controllable AAV-transgene expression are (1) highly localized to the cartilage when AAV is injected postinjury and (2) localized to the intra-articular soft tissues when AAV is injected preinjury. Two AAV injection time points, postinjury and preinjury, were investigated in osteochondral defect and anterior cruciate ligament transection models of joint injury. Rats injected with AAV tetracycline response element (TRE)-luciferase received oral doxycycline for 7 days. Luciferase expression was evaluated longitudinally for 6 months. Transgene expression was persistent and controllable with oral doxycycline for 6 months in all groups. However, the location of transgene expression was different: postinjury AAV-injected knees had luciferase expression highly localized to the cartilage, while preinjury AAV-injected knees had more widespread signal from intraarticular soft tissues. The differential transgene localization between preinjury and postinjury injection can be used to optimize treatment strategies. Highly localized postinjury injection appears advantageous for treatments targeting repair cells. The more generalized and controllable reservoir of transgene expression following AAV injection before anterior cruciate ligament transection (ACLT) suggests an intriguing concept for prophylactic delivery of joint protective factors to individuals at high risk for early osteoarthritis (OA). Successful external control of intra-articular transgene expression provides an added margin of safety for these potential clinical applications.

Gene Delivery to Joints by Intra-Articular Injection

“…Higher levels of transgene expression by synovium and cartilage were found in horses with OA (Ghivizzani et al, unpublished). These findings are consistent with prior data using rabbits (72) and rats (73) showing enhanced transduction of chondrocytes in areas of cartilage damage following intra-articular injection of AAV.…”

“…Inducible promoters have been explored as a means of regulating transgene expression in joints. Although this can be achieved with TET system (73), most research has focused on the use of self-regulating promoters that respond to inflammatory stimuli within diseased joints, as first demonstrated by Maigkov et al (80). An inducible promoter based upon a series of nuclear factor kappaB (NF-jB) response elements is entering clinical trials in RA (49,81) ( Table 1).…”

Humoral and Cell-Mediated Immune Response, and Growth Factor Synthesis After Direct Intraarticular Injection of rAAV2-IGF-I and rAAV5-IGF-I in the Equine Middle Carpal Joint

“…Intraarticular transgene expression at 56 days suggests transduction of longer lasting cell types such as chondrocytes or fibroblasts in the joint capsule and supporting ligaments, as seen in murine joints injected with AAV. 3 Although the immune response to AAV appears to be significantly less than to similar vectors, such as adenovirus, several studies have shown limited transduction efficiency and longevity of transgene expression in animals, with titers as low as 1:4. 16,34 Few studies have evaluated the immune response, and its impact on transgene expression, after direct IA injection of AAV vectors.…”

“…Previous studies have shown that articular tissues, including synoviocytes and chondrocytes, can be transduced through direct intraarticular (IA) injection. [2][3][4] Gene therapy techniques, which provide long-term in situ expression of repair-enhancing genes, would be superior to repeated injections or depots of peptide that are transient.…”

Gene Therapy in Cancer and consideration for the initiation of these therapies in Clinical Practice.

“…There is also the potential risk of genotoxicity, meaning the ability of gene therapy to cause a response in healthy unaffected cells and lead to toxicity. It is important to consider that the viral vectors may precipitate in other organs of the body such as brain or striated muscles and lead to long-term toxicity (Lee et al, 2013). The risks of gene therapy require detailed awareness of the acceptable risks, to ensure the safety of the patient, third parties who are involved, and the whole population and the environment.…”