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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.

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.