Liver-directed AAV gene therapy represents a unique treatment modality for a host of diseases. This is due, in part, to the induction of tolerance to transgene products. Despite the plethora of recognized regulatory cells in the body, there is currently a lack of literature supporting the induction of non-CD4+ regulatory cells following hepatic AAV gene transfer. In this work, we show that CD8+ regulatory T cells are up-regulated in PBMCs of mice following capsid only and therapeutic transgene AAV administration. Further, we demonstrate that hepatic AAV gene transfer results in a significant increase in CD8+ regulatory T cells following experimental autoimmune encephalomyelitis induction. Notably, this response occurred only in therapeutic vector treated animals, not capsid only controls. Understanding the role these cells play in treatment efficacy will result in the development of improved AAV vectors that take advantage of the full gamut of regulatory cells within the body.

Multiple Sclerosis is a demyelinating autoimmune disease brought on by self-reactive, myelin-specific T effector cells. Immunosuppressants are the most utilized tool to combat disease progression; however, they bring unwanted side effects. We have previously published on a novel gene immunotherapy that uses an adeno-associated virus (AAV) containing the full sequence of the myelin oligodendrocyte glycoprotein (MOG) that restores tolerance through liver-directed gene therapy, resulting in the generation of antigen-specific T regulatory cells (Tregs) that can ameliorate Experimental Autoimmune Encephalomyelitis (EAE). While the data is promising, the mechanism of this therapy remains enigmatic. To begin defining the mechanism initially, we cultured MOG-specific CD4 +T cells from 2D2 mice with liver cells from B6 mice that received either AAV.MOG or a saline control. We found a significant increase in the frequency of CD4 +CD25 +FoxP3 +Tregs when 2D2 T cells were cultured with liver cells from AAV.MOG-vectored mice in a 1:2 ratio compared to the control. Next, we injected 2D2 mice with AAV.MOG and isolated CD4 +CD25 +T cells 2 weeks later. To evaluate their suppressive ability they, or control cells, were adoptively transferred to recipient naïve B6 mice. After 24 hours, active EAE was induced. By day 15, disease severity in mice that received AAV.MOG donor 2D2 T cells was significantly reduced compared to the controls (CD4 +CD25 +T cells from wildtype B6 mice given either AAV.MOG or AAV.AQP4) Overall, these results demonstrated that livers transduced with AAV.MOG can induce 2D2 Tregs in vitro and that CD4 +CD25 +T cells from vectored 2D2 mice reduce EAE severity. Supported by a grant from the NIH (R01AI128074) and the Children's Miracle Network

In vivo induction of antigen (Ag)-specific regulatory T cells (Treg) is considered the holy grail of therapeutic strategies for restoring tolerance in autoimmunity. Unfortunately, in the autoimmune disease multiple sclerosis, an effective and durable therapy targeting the diverse repertoire of emerging Ags without compromising the patient’s natural immunity has remained elusive. To address this deficiency, we have developed an Ag-specific adeno-associated virus (AAV) immunotherapy that will restore tolerance in a Treg-dependent manner. Using multiple strains of mice with different genetic and immunological backgrounds, we demonstrate that a liver directed AAV vector expressing a single transgene can prevent experimental autoimmune encephalomyelitis from developing and effectively mitigate pre-existing or established disease that was induced by one or more auto-reactive myelin oligodendrocyte glycoprotein-derived peptides. Overall, the results suggests that AAV can efficiently restore Ag-specific immune tolerance to an immunogenic protein that is neither restricted by the major histocompatibility complex haplotype, nor by the specific antigenic epitope(s) presented. These findings may pave the way for developing a comprehensive Ag-specific immunotherapy that does not require prior knowledge of the specific immunogenic epitopes and that may prove to be universally applicable to all MS patients, and adaptable for other autoimmune diseases.

Neuromyelitis optica (NMO) is an auto-inflammatory demyelinating disease that typically affects optic nerves and spinal cord that is characterized by the presence of serum aquaporin-4 immunoglobulin G antibodies (AQP4-IgG). NMO accounts for >45% of the demyelinating disease in Asians and warrants the development of a suitable therapy other than generalized immunosuppressants. It is thought that autoimmune activated AQP4-specific T cells disrupt the BBB and allow increased entry of AQP4-IgG and other immune effectors into tissues containing astrocytes expressing AQP4 in their membranes. Recently we have established an adeno-associated viral (AAV) gene immunotherapy that effectively prevents and reverses experimental autoimmune encephalomyelitis (EAE) via generation of suppressive antigen-specific regulatory T cells (Tregs). In this report, using a similar antigen-specific approach, we have demonstrated that AAV.AQP4 gene immunotherapy prevented the development of AQP4-mediated neuroinflammation and clinical neurological disability in almost 100% of C57BL/6J mice when AAV.AQP4 vector was administered to mice 2 weeks prior to immunization with an immunogenic epitope of AQP4. Moreover, in contrast to AAV.AQP4 treated mice which remained unremarkable, histological analysis of the spinal cord sections from untreated vehicle only mice showed multiple areas of significant focal inflammation within the spinal cord. Based on our clinical and pathological data, we’ve successfully demonstrated that our novel AAV.AQP4 gene immunotherapy can indeed suppress the induction of AQP4 mediated autoimmune disease. Further evaluation will determine if it can also treat preexisting disease.

Abstract To re-establish long-term immune tolerance in Multiple Sclerosis (MS), our lab had previously developed a pre-clinical Adeno-associated virus (AAV) gene immunotherapy that is capable of preventing and reversing Myelin Oligodendrocyte Glycoprotein (MOG) induced Experimental Autoimmune Encephalomyelitis (EAE). However, MS is a disease that involves multiple myelin proteins including Proteolipid Protein (PLP). Therefore we expanded the capability of our gene immunotherapy to restore tolerance and ameliorate disease to multiple major myelin proteins, simultaneously. In this report we demonstrate that a mixture of 2 individual vectors, AAV MOG & AAV.PLP, was capable of preventing, and more importantly reversing, preexisting EAE disease induced with a mixture of MOG35–55 & PLP139–151 peptides in (C57BL/6JxSJL)F1 mice. To minimize the total vector load we further developed this immunotherapy into a novel single AAV-dual transgene expressing vector (AAV.MOG.PLP) using specific gene linkers for independent expression. Following a single peripheral injection, western blot analysis confirmed stable and simultaneous hepatic expression of both neuroprotein transgenes. When injected into mice 2 weeks before induction of EAE with either MOG35–55 or PLP139–151, or combination of both, the dual transgene AAV immunotherapy significantly reduced or prevented disease in mice compared to controls. Overall, these results demonstrate proof of concept that a single AAV vector simultaneously expressing more than one transgene may be an effective therapeutic treatment for restoring tolerance in an autoimmune disease like MS. Supported by NIHSarepta Pharmaceuticals NIH R01Act AI128074Project

Adeno-associated virus (AAV) vectors are widely used in clinical gene therapy to correct genetic disease by in vivo gene transfer. Although the vectors are useful, in part because of their limited immunogenicity, immune responses directed at vector components have complicated applications in humans. These include, for instance, innate immune sensing of vector components by plasmacytoid dendritic cells (pDCs), which sense the vector DNA genome via Toll-like receptor 9. Adaptive immune responses employ antigen presentation by conventional dendritic cells (cDCs), which leads to cross-priming of capsid-specific CD8+ T cells. In this study, we sought to determine the mechanisms that promote licensing of cDCs, which is requisite for CD8+ T cell activation. Blockage of type 1 interferon (T1 IFN) signaling by monoclonal antibody therapy prevented cross-priming. Furthermore, experiments in cell-type-restricted knockout mice showed a specific requirement for the receptor for T1 IFN (IFNaR) in cDCs. In contrast, natural killer (NK) cells are not needed, indicating a direct rather than indirect effect of T1 IFN on cDCs. In addition, co-stimulation by CD4(+) T cells via CD40-CD40L was required for cross-priming, and blockage of co-stimulation but not of T1 IFN additionally reduced antibody formation against capsid. These mechanistic insights inform the development of targeted immune interventions.