In recent years there has been tremendous progress in the search for biological solutions to the problem of disc degeneration. Gene therapy has emerged as a promising therapeutic option with tremendous potential, and while researchers continue to study its efficacy, it is incumbent upon those seeking clinical application to fully evaluate its considerable risk profile. This experiment was done in the same vein as the safety study by Wallach et al, which demonstrated that while properly dosed and delivered gene therapy can effect dramatic therapeutic change within the IVD, misplacement or incorrect dosing can lead to devastating side effects. Just as the previous study examined effects of subarachnoid
placement, our study sought to mimic misplacement or leakage of recombinant constructs into the epidural
space, a complication known to occur during other intradiscal procedures such as discography [23
]. In addition, by comparing adenoviral and adeno-associated viral vectors, we sought to determine the role of vector choice in post-injection morbidity.
Prior to embarking on this study, we hypothesized that placement of genetic constructs into the epidural space would not result in extensive morbidity. Being that the epidural space is highly vascular, we postulated that the circulation would “wash away” injected material before the vector had an opportunity to transducer any cells, a theory that has been discussed in gene therapy literature [30
]. Also the injection would be placed outside of the dura mater, separated from the spinal cord by the blood-brain barrier, limiting its ability to cause pathology within the spinal cord.
Several conclusions might be drawn from the somewhat surprising results from the adenoviral arm of our study. First, it appears that adenovirus is an extremely aggressive vector, capable of rapid and vigorous transduction of cells both in proximity and at a distance to the original site of its insertion. The presence of marker gene expression within the parenchyma of the spinal cord in our study supports the notion that adenovirus is capable of crossing the blood-brain barrier. That marker gene product appears exclusively within cells suggests that the viral particles themselves are taken in by cells, rather than simply transport of marker protein from remote sites. Transport of proteins would have likely resulted in the observance of marker gene in the extracellular matrix, which we did not see. While the precise mechanism by which the barrier is breached is beyond the scope of this paper, prior reports have noted an ability of viral vectors to gain access to the central nervous system via a retrograde axonal transport mechanism [24
]. Our study perhaps lends support to this theory, and in addition suggests that adenoviral vectors are better able than AAV vectors to access the central nervous system from points of insertion outside the blood-brain barrier. Second, our study suggests that it is necessary for there to be a critical overdose of gene therapy outside of the disc to cause deleterious effects, as very little clinical or histological pathology was detected in any of the rabbits receiving a physiologic dose of Ad/BMP-2. As prior work has shown a 106
pfu dose to be sufficient in effecting therapeutic change in disc cells [25
], our study suggests that an entire therapeutic dose of gene therapy can be misplaced into the epidural space without causing any clinical morbidity. Certainly this optimism must be tempered though, as very serious side effects were seen in the majority of rabbits receiving higher doses of Ad/TGF-β1 and Ad/BMP-2, indicating that there exists a definite therapeutic index and highlighting the importance of proper dosing.
The paper by Wallach et al mentioned three potential mechanisms potentially responsible for the observed clinical morbidity. Our current study lends support to their conclusion that clinical and histological pathology is caused by a synergism between transgene product and viral vector. Five of six rabbits receiving a supratherapeutic dose of BMP-2 via an adenoviral vector suffered severe neurological problems, yet rabbits receiving a supratherapeutic dose of the same transgene via an AAV vector had no significant complications. Based on our current study, we suggest two ways in which the vector and the transgene combine to cause subject morbidity at both the macroscopic and cellular levels. First, our data indicate that an increased level of certain biologically active transgene products correlates with increased morbidity. Indeed, while a high dose of Ad-LacZ resulted only in a small amount of histological inflammation, the same dose of Ad-BMP-2 resulted in serious adverse effects. Yet our data also suggests that these effects cannot manifest unless the viral vector is capable of delivering this transgene to the tissues. As we observed, high doses of adenoviral constructs caused devastating side effects, while high doses of AAV constructs carrying the same transgene (BMP-2) resulted in neither clinical nor histological morbidity whatsoever. Though the exact pharmacokinetic profile of these vectors is not known and is beyond the scope of this study, it appears that AAV may be a less aggressive vector than adenovirus, limiting its transduction efficiency in highly vascular areas such as the epidural space. Indeed, there is evidence in the literature to support this concept. A study done on New Zealand white rabbits looking at transgene expression following delivery by AAV and adenovirus to the jugular veins demonstrated a significantly more efficient response with an adenoviral carrier [26
]. In our study, while extensive expression of marker gene occurred in the tissues of rabbits injected with adenoviral constructs, there was no marker transgene expression following delivery via AAV (). We therefore suspect that while dilution by the epidural circulation may have prevented transduction of the surrounding cells by AAV, the more aggressive adenoviral vector was still able to function, and the consequences of supratherepeutic dosing was able to manifest. The variable transduction efficiency AAV demonstrates between different physiologic compartments in the body may point to a significant safety advantage whereby any vector delivered outside of the hypovascular target tissue (i.e. nucleus pulposus) is effectively neutralized by the hypervascularity of the surrounding tissues.
Second, we submit that the decreased antigenicity of AAV may help to limit the host immune response and lessen the damage done by lymphocytic infiltration into the areas of vector uptake. There is an abundance of literature that describes the differences in the immune response to adenoviral and adeno-associated viral vectors, and it seems that, particularly with respect to cell-mediated immune reactions, the response to AAV is considerably less aggressive [27
]. Clearly, as evidenced by the low levels of inflammation caused by high dose Ad-LacZ, the viral vector of choice is not likely to be the primary cause of deleterious effects. It should be noted however that we observed no histological evidence of inflammation whatsoever in the AAV arm of the study. Future studies involving these types of injections should examine typical circulatory depots, including the liver and lymphoid tissue, for any evidence of vector uptake.
While we have learned much from this project, it does have certain limitations. First, the total number of subjects in our study is small, owing to our attempt to limit the overall morbidity of our pilot study. While the lack of any adverse side effects in any of our AAV subjects would suggest that AAV is a safe vector, a direct comparison between it and adenovirus was limited by the available titer of our AAV vector. Unfortunately we were able to increase our dose of AAV by a factor of ten, compared to a 100-fold increase for adenovirus, and it would be reasonable to question whether we achieved a toxic dose of either AAV or the transgene product in our high dose AAV groups. Finally, with our study only spanning six weeks, it is possible that we are missing some later effects of the injections, particularly in those rabbits receiving AAV constructs. Future studies should be directed toward examining the potential long-term effects of misplaced or improperly dosed gene therapy.
In summary, gene therapy holds great potential to become a viable option in the biological treatment of intervertebral disc degeneration. While there do seem to be substantial benefits to the use of gene therapy, it has become clear that these do not come free of patient risk, and an increasing number of projects are being directed toward finding safer means by which to implement the technology [31
]. Our current study seeks to shed some light not only on the benefits of genetic modulation, but also on the challenges it may pose when used to treat conditions of the spine. Prior to widespread clinical application, a precise therapeutic dose must be determined, as well as the best possible means by which to deliver this dose. In addition, we must develop a feasible method to consistently ensure the appropriate and accurate delivery of vector-gene constructs to the disc space. With persistent attention to issues of safety, research regarding the use of gene therapy will continue to progress, bringing us closer to its clinical application to the problem of intervertebral disc degeneration.