In the present study, we evaluated patterns of transgene expression in articular cells after infection with scAAV vectors, first in vitro
and then in vivo
in normal and inflamed joints. We found that, in culture, the scAAV vector represented a significant technical advance over conventional single-stranded vectors with regard to cellular transduction, providing an approximately 25-fold enhancement in transgenic expression. The comparatively poor performance of the conventional vector indicates that second-strand DNA synthesis can be a major impediment to effective transduction of joint tissues. Concerning normal and inflamed articular environments, we found no significant difference in the levels or duration of expression of the IL-1Ra transgene after delivery of the self-complementary vector. Generally, after injection of approximately 5 × 1011
particles, sufficient levels of IL-1Ra transgene product were generated to cause a reduction in the leukocytic infiltration in joints inflamed by constitutive IL-1 production. By contrast to previous studies [39
], we found that, after the loss of IL-1Ra transgene expression, neither re-injection of the scAAV.IL-1Ra vector, nor the induction of a second inflammatory response could generate detectable levels of IL-1Ra expression intra-articularly.
In animal studies of intra-articular transgene expression, we have found several advantages to the rabbit knee as a model system. Being approximately the same size as the metacarpophalangeal joints of the human hand, a frequent site of rheumatoid arthritis, it offers a reasonable simulation of the process of gene delivery in the treatment of human joint disease. Given that the studies published to date have been performed in rats and mice [17
] with joints being 1–2 log orders smaller than those in humans, we believe the data obtained in the present study are among the first to report intra-articular transgene expression from an AAV vector in articular tissues on a clinically applicable scale. As shown by the ability of the IL-1Ra expression to alleviate leukocytosis in inflamed joints of the rabbits, the efficiency of scAAV-mediated gene delivery and ensuing expression is sufficient to induce a beneficial biological response in this context.
The patterns of transgene expression observed with the self-complementary vector in rabbits differ somewhat from those observed in the joints of mice injected with conventional AAV. In the murine system, AAV mediated transgene expression was found to onset significantly earlier in arthritic joints, and levels of expression were greater than in normal joints [17
]. This was primarily attributed to differences in the synthesis of the second DNA strand of the AAV vector within the infected cell, and increased production of DNA synthesis/repair enzymes in cells receiving inflammatory stimuli. With the self-complementary vector, AAV-mediated transgene expression from normal rabbit synovial fibroblasts in culture as well as in vivo
in normal joints had a rapid onset, with no evidence of delay relative to arthritic joints. Thus, the self-complementary vector bypasses the variability associated with conventional AAV vectors in the arthritic environment, and thereby provides a more predictable gene delivery reagent. The observation that cells within both normal and inflamed joints of the rabbit are equally capable of being transduced by an scAAV-based vector indicates that this system may have application in a spectrum of articular ailments. These include inflammatory conditions, such as rheumatoid arthritis, as well as those not directly associated with chronic inflammation, such as osteoarthritis, and repair of joint tissues, such as meniscus and ligament.
Although intra-articular gene transfer accompanied by limited inflammation has been previously reported with AAV, the capacity of this vector to enable persistent transgenic expression in joint tissues has yet to be fully assessed. In the present study, we found that, within the rabbit knee, expression of the human IL-1Ra transgene was gradually lost over a period of a few weeks and that re-administration of the vector could not restore expression. Unfortunately, the human IL-1Ra transgene product used in these experiments, although extremely useful as a secretable marker, is at the same time immunogenic when administered across species boundaries to the joints of normal, immunocompetent animals [15
]. We have found similar patterns of abbreviated intra-articular expression after the use of other xenogenic transgene products, regardless of whether they are secreted or intracellular [15
]. Recent studies have shown that, in the absence of specific T-cell mediated immunity directed against nonself proteins of transgenic or viral vector origin, cells within fibrous tissues of the joint can support long-term (> 6 months) transgenic expression [15
]. The capacity with which AAV vectors can infect and transduce these particular cell types is currently unknown, but is an area of ongoing study within our group, as well as others.
AAV-mediated transduction of the target cell involves several key steps that broadly include viral attachment and entry [43
], intracellular trafficking to the nucleus [45
], nuclear entry and uncoating [48
], and conversion of the single-stranded genome into a double-stranded form [50
]. In attempting to improve the efficiency of AAV transduction, the development of methods to cross-package vector genomes in alternate capsid serotypes has dramatically expanded the host cell range of the widely used, AAV serotype 2-based vectors. Furthermore, the development of scAAV vectors bypasses the limitations associated with second-strand DNA synthesis. As shown in the present study, as well as those conducted previously, important barriers to transduction of articular fibroblasts appear to still remain at the level of intracellular trafficking [31
]. Relative to other viral vectors, high numbers of viral particles are required to achieve transduction of human, rat and rabbit synovial fibroblasts in culture, typically in the range of 104
viral particles per cell and, as shown in the present study, only between 10% and 20% of AAV genomes enter the nucleus. Several lines of evidence implicate the ubiquitin-proteasome pathway as a key hurdle to efficient intracellular trafficking by AAV2-based vectors and other serotypes. Jennings et al
] showed that the addition of proteasome inhibitors, such as carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (zLLL), dramatically enhanced nuclear uptake of AAV genomes in human synovial fibroblasts and was accompanied by a proportional increase in transgenic expression. More recently, Zhong et al
] demonstrated that cellular phosphorylation of specific tyrosine residues on the AAV capsid surface led to increased ubiquitination of the viral particle and enhanced proteasome degradation. Site-directed mutagenesis of these tyrosines to phenylalanine blocked ubiquitination and led to an approximately ten-fold enhancement of transduction efficiency [55
]. The relative utility of these modified capsids in articular cells has not been investigated but, similar to the self-complementary vectors, they have the potential to significantly enhance the efficiency of AAV-mediated gene transfer.