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We have reported that of the 10 most commonly used adeno-associated virus (AAV) serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs) in vitro, as well as in vivo. More recently, polyvinyl alcohol (PVA), was reported to be a superior replacement for human serum albumin (HSA) for ex vivo expansion of HSCs. Since HSA has been shown to increase the transduction efficiency of AAV serotype vectors, we evaluated whether PVA could also enhance the transduction efficiency of AAV6 vectors in primary human HSCs. We report here that up to 12-fold enhancement in the transduction efficiency of AAV6 vectors can be achieved in primary human HSCs with PVA. We also demonstrate that the improvement in the transduction efficiency is due to PVA-mediated improved entry and intracellular trafficking of AAV6 vectors in human hematopoietic cells in vitro, as well as in murine hepatocytes in vivo. Taken together, our studies suggest that the use of PVA is an attractive strategy to further improve the efficacy of AAV6 vectors. This has important implications in the optimal use of these vectors in the potential gene therapy and genome editing for human hemoglobinopathies such as β-thalassemia and sickle cell disease.

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.

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.

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 viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in “humanized” mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.

The devastating neurodegenerative disease multiple sclerosis (MS) could substantially benefit from an adeno-associated virus (AAV) immunotherapy designed to restore a robust and durable antigen-specific tolerance. However, developing a sufficiently potent and lasting immune-regulatory therapy that can intervene in ongoing disease is a major challenge and has thus been elusive. We addressed this problem by developing a highly effective and robust tolerance-inducing in vivo gene therapy. Using a pre-clinical animal model, we designed a liver-targeting gene transfer vector that expresses full-length myelin oligodendrocyte glycoprotein (MOG) in hepatocytes. We show that by harnessing the tolerogenic nature of the liver, this powerful gene immunotherapy restores immune tolerance by inducing functional MOG-specific regulatory T cells (Tregs) in vivo, independent of major histocompatibility complex (MHC) restrictions. We demonstrate that mice treated prophylactically are protected from developing disease and neurological deficits. More importantly, we demonstrate that when given to mice with preexisting disease, ranging from mild neurological deficits to severe paralysis, the gene immunotherapy abrogated CNS inflammation and significantly reversed clinical symptoms of disease. This specialized approach for inducing antigen-specific immune tolerance has significant therapeutic potential for treating MS and other autoimmune disorders., Graphical Abstract, Multiple sclerosis is a neurodegenerative autoimmune disease with no known cure. Hoffman and colleagues present an AAV-based immunotherapy that induces antigen-specific tolerance to a neuropeptide that not only prevents disease onset, but is robust enough to alleviate preexisting neuro-inflammation and reverse disease in a model of MS.

Microdialysis sampling is a commonly used technique for collecting solutes from the extracellular space of tissues in laboratory animals and humans. Large molecular weight solutes can be collected using high molecular weight cutoff (MWCO) membranes (100 kDa or greater). High MWCO membranes require addition of high molecular weight dextrans or albumin to the perfusion fluid to prevent fluid loss via ultrafiltration. While these perfusion fluid additives are commonly used during microdialysis sampling, the tissue response to the loss of these compounds across the membrane is poorly understood. Tissue reactions to implanted microdialysis sampling probes containing different microdialysis perfusion fluids were compared over a 7-day time period in rats. The base perfusion fluid was Ringer’s solution supplemented with either bovine serum albumin (BSA), rat serum albumin (RSA), Dextran-70, or Dextran-500. A significant inflammatory response to Dextran-70 was observed. No differences in the tissue response between BSA and RSA were observed. Among these agents, the BSA, RSA, and Dextran-500 produced a significantly reduced inflammatory response compared to the Dextran-70. This work demonstrates that use of Dextran-70 in microdialysis sampling perfusion fluids should be eliminated and replaced with Dextran-500 or other alternatives.

Immune tolerance is a vital component of immunity, as persistent activation of immune cells causes significant tissue damage and loss of tolerance leads to autoimmunity. Likewise, unwanted immune responses can occur in inherited disorders, such as hemophilia and Pompe disease, in which patients lack any expression of protein, during treatment with enzyme replacement therapy, or gene therapy. While the liver has long been known as being tolerogenic, it was only recently appreciated in the last decade that liver directed adeno-associated virus (AAV) gene therapy can induce systemic tolerance to a transgene. In this review, we look at the mechanisms behind liver induced tolerance, discuss different factors influencing successful tolerance induction with AAV, and applications where AAV mediated tolerance may be helpful.

Similar to Multiple Sclerosis, EAE is a complex disease in which immune tolerance is lost and antigen specific (Ag-sp) Tregs are known to play a role. We have developed a gene immunotherapy that has been shown to induce MOG specific Tregs, restore tolerance, and effectively prevent, and/or reverse, significant disease. However, the spatiotemporal profile of the induced Tregs is unclear. To identify the tissue specific populations of AAV8.MOG-induced specific Tregs, we used a transgenic Foxp3 reporter mouse with a MOG35–55/I-Ab tetramer. Two weeks prior to EAE induction, mice were tolerized via a single injection of vector. At the peak of disease, tissues were harvested and fluorescently labeled for phenotypic identification. FACS analysis revealed a higher proportion of Ag-sp Tregs in the cervical and inguinal lymph nodes (LN) and spleen, as compared to controls. In contrast, a higher proportion of Ag-sp effector cells were seen in control mice as compared to treated. Furthermore, a significant number of infiltrating effector T cells were isolated from the spinal cords of control mice, while very few T cells were recovered from treated mice. This aligns with cytokine data that was collected showing there to be higher production of IFN-γ in the spinal cord and spleen of control mice. These results suggest that AAV.MOG induced Ag-sp Treg populations accumulate in the peripheral organs, and may prevent effector cells from migrating to the spinal cord. This work provides new insights on tolerance induction mechanisms. These insights will be useful for the development of effective cell based therapies capable of treating autoimmune diseases.

Autoimmune disease is caused by a breakdown of tolerance against endogenous protein(s). New therapies are being developed that utilize the natural abilities of regulatory T (Treg) cells to suppress autoimmune effector cells in an antigen-specific manner. Recently, we demonstrated a clinically relevant in vivo gene immunotherapy approach for restoring Ag-specific tolerance using hepatic adeno-associated virus (AAV) gene transfer. This approach was shown to be remarkably successful at preventing and reversing a monophasic, MOG-induced EAE disease. Additionally, we have shown the dynamic ability of this treatment to prevent and/or reverse disease induced with various MOG epitopes, in genetically diverse mouse strains. However, it is unclear if this approach would be effective in the more complicated, PLP induced relapsing-remitting disease model. Thus, we re-engineered our therapeutic vector to contain the full-coding sequence of PLP. This is critically important as the established RR-EAE (SJL) model is characterized by epitope spreading; a phenomenon that may more closely resemble multiple sclerosis. SJL mice received a single peripheral injection of AAV. PLP vector, or control, 2 weeks prior to PLP 139–151 induced EAE. By day 14, control mice developed significant neurological deficits followed by a relapsing-remitting disease course. In contrast, the treated mice failed to exhibit any neurological symptoms of disease throughout the duration of the experiment, ~75 days. The long-term prevention of PLP induced disease, provides the first evidence of an effective Treg based therapy for combating epitope drift in RR-EAE, and potentially a new paradigm for treating the autoimmune disease multiple sclerosis.

Macrophage activation is of interest in the biomaterials field since macrophages with an M(Dex) characteristic phenotype, i.e., CD68 + CD163 + , are believed to result in improved integration of the biomaterial as well as improved tissue remodeling and increased biomaterial longevity. To facilitate delivery of a macrophage modulator, dexamethasone-21-phosphate (Dex), microdialysis probes were subcutaneously implanted in male Sprague–Dawley rats. Dex localized delivery was delayed to the third day post implantation as a means to alter macrophage activation state at an implant site. To better elucidate the molecular mechanisms associated with M(Dex) macrophage activation, CCL2 was quantified in dialysates, gene expression ratios were determined from excised tissue surrounding the implant, histological analyses, and immunohistochemical analyses (CD68, CD163) were performed. Delayed Dex infusion resulted in the up-regulation of IL-6 at the transcript level in the tissue in contact with the microdialysis probe and decreased CCL2 concentrations collected in dialysates. Histological analyses showed increased cellular density as compared to controls in response to delayed Dex infusion. Dex delayed infusion resulted in an increased percentage of CD68 + CD163 + , M(Dex), macrophages in the tissue surrounding the microdialysis probe as compared to probes that served as controls.

Cell-based immunotherapies have the potential to reverse autoimmune disease by inducing or restoring immune-tolerance. Extensive preclinical studies have demonstrated that regulatory T cell (Tregs) play a key role in both induction and maintenance of tolerance. Several strategies aimed at harvesting and expanding autologous Tregs and then re-infusing them into patients have been investigated. Unfortunately, development of a large scale in vitro method has not been achieved, especially for human cells. On the other hand, a clinically relevant in vivo approach for inducing Ag-specific tolerance has successfully been demonstrated using ectopic expression of an antigen in the liver. Here, we have developed a long-lasting immunotherapy that induces functionally suppressive, Ag-specific Tregs in the murine model of multiple sclerosis using a liver directed adeno-associated virus (AAV) vector expressing myelin oligodendrocyte glycoprotein (MOG). Modulation of EAE disease in C57BL/6 is typically limited to a single immunodominant epitope. By engineering the AAV vector to express full-length protein (MOG1-247), we have created an immunotherapy that is capable of dynamically inducing Ag-specific tolerance against multiple immunogenic epitopes (e.g MOG35-55, MOG119-132). Mice received a single peripheral injection of AAV.MOG vector, or control. EAE was induced between 2 weeks and 8 months later using various MOG epitopes emulsified in adjuvant. Within 14-days, control mice developed significant neurological deficits, whereas none of the treated mice exhibited any symptoms. This work completely abrogates the deficiencies associated with ex vivo expanded Tregs providing a new paradigm for treating autoimmune diseases.

Microdialysis sampling probes were implanted into the subcutaneous space on the dorsal side of male Sprague Dawley rats to locally deliver dexamethasone-21-phosphate (Dex) with the aim of altering in vivo macrophage polarization. Macrophage polarization is of significant interest in the field of biomaterials since wound-healing macrophages are a possible means to extend implant life as well as improve tissue remodeling to an implant. Quantitative analysis of CCL2 in collected dialysates, gene expression and immunohistochemistry performed on the tissue surrounding the microdialysis implant were used to evaluate if Dex polarized macrophages. Dex infusion down-regulated IL-6 and CCL2 gene expression and decreased CCL2 concentrations in dialysates collected at the implant site. Dex appeared to have no significant effect on the gene regulation of CD163, a commonly used M2c macrophage surface marker; Arg2; and iNOS2. However, Dex infusion was effective at increasing the number of CD163+ cells surrounding the implanted microdialysis probe. This work demonstrates the use of microdialysis sampling to deliver agents such as Dex to alter macrophage polarization in vivo while allowing the ability to collect cytokines in the surrounding microenvironment.

Induction of peripheral immune tolerance requires regulatory T cells (Tregs). Protocols designed to manipulate antigen (Ag) specific-Treg populations in vivo, represent a powerful approach for treating autoimmune disease such as Multiple Sclerosis (MS). It has been shown that Ag-specific Tregs can play a critical role in the protection and recovery of MS, and its animal model, experimental autoimmune encephalomyelitis (EAE). Unfortunately, methods to induce endogenous CNS-targeting Ag-specific Tregs that achieve therapeutic efficacy are lacking. To address this, we developed an immune tolerance induction therapy using an AAV vector expressing the full coding sequence for the neuro-protein MOG under the control of a strong liver specific promoter. Our data demonstrates that peripheral injection of this vector induces functionally suppressive transgene-specific Tregs, and mice pre-treated with this vector are protected from developing EAE. More notably, when vector is administered during mild to moderate disease, there is a nearly complete reversal of EAE, both clinically and histopathologically. When treatment is withheld until mice exhibit moderately severe EAE, our therapy still significantly reduced the neurological symptoms and clinical score, although to a lesser degree. Thus, to increase efficacy of the vector induced Tregs, a short course of rapamycin treatment was given concomitantly which had a synergistic affect, resulting in a long-term significant reversal of disease. Using our vector platform to deliver full-length proteins offers a superior MHC/HLA independent approach for in vivo induction of Ag-specific Tregs compared to other ex vivo or epitope restricted Treg mediated therapies being investigated.