rAAV vectors expressing a variety of transgenes have been used successfully in the treatment of a multitude of diseases, including T1D (1-5). In this study, we have shown that administration of a rAAV vector expressing IL-2 under control of a β cell specific promoter, mIP (AAV8mIP-IL2), was able to prevent diabetes in NOD mice (Chapter 3). This protection was mediated by specific effects on islet resident FoxP3+CD25+ Treg. Furthermore, in remission studies, AAV8mIP-IL2 vaccination was also shown to work synergistically with the non-depleting αCD4 and αCD8 monoclonal antibodies, YTS177 and YTS105.
Mechanistically, the beneficial effects of AAV8mIP-IL2 on long term YTS treated NOD mice do not appear to be limited strictly to effects on FoxP3+ Treg. However, in prediabetic NOD mice treated with both AAV8mIP-IL2 and the YTS antibodies short term, the frequency of FoxP3+ Treg in the islets was significantly enhanced compared to YTS-only recipients. This would suggest that AAV8mIP-IL2 can specifically counteract the loss of FoxP3+ Treg from organs of interest, notably the islets, after YTS treatment. By maintaining a larger frequency of FoxP3+ Treg in the islets, the beneficial effects of YTS
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mediated immunotherapy would be enhanced. Given that systemic IL-2 levels were stably maintained in long term remission animals suggests that any effects on islet FoxP3+ Treg would be maintained throughout the course of remission. Therefore, since investigations concerning islet FoxP3+ Treg in long term YTS-only treated animals is lacking, comparisons between combinatorial AAV8mIP-IL2 and YTS and YTS-only long term treated remission NOD mice merit consideration.
The dosing of IL-2 in vivo was shown to be intrinsic to therapeutic function in both NOD mice and diabetic human patients. Investigations utilizing both prediabetic and recent onset diabetic NOD mice have shown that low dose rIL-2 therapy acts specifically on FoxP3+ Treg to induce protection and/or remission (6-8). Importantly, high dose administration resulted in significant off target effects, thereby eliminating any potential benefits of treatment (8). Similarly, a study utilizing rIL-2 combinatorial therapy in recent onset human patients showed expansion of both NK cells and
eosinophils, thereby reducing its clinical efficacy, despite modest increases in systemic FoxP3+ Treg (9). This would suggest that dose is of particular importance for
translational approaches.
Although long term systemic transgene levels were low and stable throughout the lifespan of AAV8mIP-IL2 treated NOD mice, constant and increased IL-2 levels could potentially have long term adverse effects on the immune system. We have previously shown that a rAAV vector tetracycline inducible (TET-ON) system expressing IL-2 also successfully prevented diabetes in NOD mice, despite a short 3 week induction phase
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(2). Both the β cell specific (mIP) and inducible (Tet-ON) rAAV vectors offer distinct advantages over the other. As such, the use of a dual promoter rAAV vector that is both β cell-specific and inducible would be attractive. Development of this vector would allow for localized, short term bursts of transgene to the islets, while significantly curtailing both the magnitude and length of systemic exposure. We recently packaged a rAAV8 vector expressing IL-2 under the control of both the TET-ON promoter and mIP (dual promoter IL-2), which was shown to secrete IL-2 transgene in vitro and increase the frequency of pancreas FoxP3+ Treg in vivo (M.C.J. and R.T., unpublished results). Many questions concerning vector dosage and route of administration, systemic versus localized expression of transgene, and the overall effect on diabetes incidence need to be investigated. However, the availability of an immunotherapy that is both targeted and controllable provides an interesting platform for future immunotherapy
approaches.
Vaccination with pDNA expressing β cell autoantigens results in the rapid
expansion of β cell specific FoxP3+ Treg (10-12). Increases in antigen-specific FoxP3+ Treg can prevent diabetes in young NOD mice, but efficacy wanes upon treatment of older animals. Along these lines, we have shown that combinatorial therapy with AAV8mIP- IL2 and pDNA encoding the β cell autoantigen glutamic acid decarboxylase 65 (GAD65) increased the frequency of GAD65 p217-specific FoxP3+ Treg in the islets of treated NOD mice (data not shown). The ability of this combinatorial approach to prevent the onset of T1D, particularly in older NOD mice, however, was not investigated. Given the success of AAV8mIP-IL2 therapy alone in preventing diabetes in NOD mice treated with a “high”
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dose of vector, it would be of particular interest to investigate the efficacy of this strategy with suboptimal levels of AAV8mIP-IL2.
The advent of our dual promoter system raises a novel avenue for the investigation of combinatorial approaches to treat T1D. Multiple β cell autoantigens have been identified as playing a role in the progression of T1D, many of which have been investigated in the context of pDNA vaccination (13-16). By staggering treatment of NOD mice with pDNA encoding different β cell autoantigens, and subsequently coupling the induction of our dual promoter IL-2 system with administration of those different pDNA, the expansion of various antigen-specific FoxP3+ Treg populations can occur. Potentially, this would establish a substantial pool of protective, β cell specific FoxP3+Treg specifically in the islets, that in turn would be expected to overcome some of the limitations associated with pDNA vaccination.