Upregulation of CD8+ regulatory T cells following liver-directed AAV gene therapy.

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.

AAV3-miRNA vectors for growth suppression of human hepatocellular carcinoma cells in vitro and human liver tumors in a murine xenograft model in vivo.

We have previously reported that recombinant adeno-associated virus serotype 3 (AAV3) vectors transduce human liver tumors more efficiently in a mouse xenograft model following systemic administration. Others have utilized AAV8 vectors expressing miR-26a and miR-122 to achieve near total inhibition of growth of mouse liver tumors. Since AAV3 vectors transduce human hepatic cells more efficiently than AAV8 vectors, in the present studies, we wished to evaluate the efficacy of AAV3-miR-26a/122 vectors in suppressing the growth of human hepatocellular carcinoma (HCC) cells in vitro, and human liver tumors in a mouse model in vivo. To this end, a human HCC cell line, Huh7, was transduced with various multiplicities of infection (MOIs) of AAV3-miR-26a or scAAV3-miR-122 vectors, or both, which also co-expressed a Gaussia luciferase (GLuc) reporter gene. Only a modest level of dose-dependent growth inhibition of Huh7 cells (~12-13%) was observed at the highest MOI (1 × 105 vgs/cell) with each vector. When Huh7 cells were co-transduced with both vectors, the extent of growth inhibition was additive (~26%). However, AAV3-miR-26a and scAAV3-miR-122 vectors led to ~70% inhibition of growth of Huh-derived human liver tumors in a mouse xenograft model in vivo. Thus, the combined use of miR-26a and scAAV3-miR-122 delivered by AAV3 vectors offers a potentially useful approach to target human liver tumors.

Type I IFN Sensing by cDCs and CD4+ T Cell Help Are Both Requisite for Cross-Priming of AAV Capsid-Specific CD8+ T Cells.

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.

Liver induced transgene tolerance with AAV vectors.

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.

Regulatory T cells and TLR9 activation shape antibody formation to a secreted transgene product in AAV muscle gene transfer.

Adeno-associated viral (AAV) gene delivery to skeletal muscle is being explored for systemic delivery of therapeutic proteins. To better understand the signals that govern antibody formation against secreted transgene products in this approach, we administered an intramuscular dose of AAV1 vector expressing human coagulation factor IX (hFIX), which does not cause antibody formation against hFIX in C57BL/6 mice. Interestingly, co-administration of a TLR9 agonist (CpG-deoxyoligonucleotide, ODN) but not of lipopolysaccharide, caused a transient anti-hFIX response. ODN activated monocyte-derived dendritic cells and enhanced T follicular helper cell responses. While depletion of regulatory T cells (Tregs) also caused an antibody response, TLR9 activation combined with Treg depletion instead resulted in prolonged CD8+ T cell infiltration of transduced muscle. Thus, Tregs modulate the response to the TLR9 agonist. Further, Treg re-population eventually resolved humoral and cellular immune responses. Therefore, specific modes of TLR9 activation and Tregs orchestrate antibody formation in muscle gene transfer.

Plasmacytoid and conventional dendritic cells cooperate in crosspriming AAV capsid-specific CD8+ T cells.

Adeno-associated virus (AAV) is a replication-deficient parvovirus that is extensively used as a gene therapy vector. CD8+ T-cell responses against the AAV capsid protein can, however, affect therapeutic efficacy. Little is known about the in vivo mechanism that leads to the crosspriming of CD8+ T cells against the input viral capsid antigen. In this study, we report that the Toll-like receptor 9 (TLR9)-MyD88 pattern-recognition receptor pathway is uniquely capable of initiating this response. By contrast, the absence of TLR2, STING, or the addition of TLR4 agonist has no effect. Surprisingly, both conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) are required for the crosspriming of capsid-specific CD8+ T cells, whereas other antigen-presenting cells are not involved. TLR9 signaling is specifically essential in pDCs but not in cDCs, indicating that sensing of the viral genome by pDCs activates cDCs in trans to cross-present capsid antigen during CD8+ T-cell activation. Cross-presentation and crosspriming depend not only on TLR9, but also on interferon type I signaling, and both mechanisms can be inhibited by administering specific molecules to prevent induction of capsid-specific CD8+ T cells. Thus, these outcomes directly point to therapeutic interventions and demonstrate that innate immune blockade can eliminate unwanted immune responses in gene therapy.

Gene Therapy-Induced Antigen-Specific Tregs Inhibit Neuro-inflammation and Reverse Disease in a Mouse Model of Multiple Sclerosis.

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.

The Balance between CD8+ T Cell-Mediated Clearance of AAV-Encoded Antigen in the Liver and Tolerance Is Dependent on the Vector Dose.

The liver continuously receives antigens from circulation and the gastrointestinal tract. A complex immune regulatory system has evolved in order to both limit inflammation and promote tolerance in the liver. Although in situ immune tolerance mechanisms enable successful gene therapy and liver transplantation, at the same time they facilitate chronic infections by pathogens such as hepatitis viruses. It is, however, poorly understood why hepatocytes infected with hepatitis viruses or transduced with adeno-associated virus (AAV)-based vectors may be rejected by CD8+ T cells several months later. We found that hepatic transfer of limited doses of an AAV-ovalbumin vector rapidly induced antigen-specific CD8+ T cells that only became functionally competent after >2 months. At this time, CD8+ T cells had downregulated negative checkpoint markers, e.g., the programmed death 1 [PD-1] receptor, and upregulated expression of relevant cytokines. At further reduced vector dose, only intrahepatic rather than systemic CD8+ T cell responses occurred, showing identical delay in antigen clearance. In contrast, PD-1-deficient mice rapidly cleared ovalbumin. Interestingly, higher vector dose directed sustained transgene expression without CD8+ T cell responses. Regulatory T cells, IL-10 expression, and Fas-L contributed to high-dose tolerance. Thus, viral vector doses profoundly impact CD8+ T cell responses.

Alpha-1 Antitrypsin-Deficient Macrophages Have Increased Matriptase-Mediated Proteolytic Activity.

Alpha-1 antitrypsin (AAT) deficiency-associated emphysema is largely attributed to insufficient inhibition of neutrophil elastase released from neutrophils. Correcting AAT levels using augmentation therapy only slows disease progression, and that suggests a more complex process of lung destruction. Because alveolar macrophages (Mɸ) express AAT, we propose that the expression and intracellular accumulation of mutated Z-AAT (the most common mutation) compromises Mɸ function and contributes to emphysema development. Extracellular matrix (ECM) degradation is a hallmark of emphysema pathology. In this study, Mɸ from individuals with Z-AAT (Z-Mɸ) have greater proteolytic activity on ECM than do normal Mɸ. This abnormal Z-Mɸ activity is not abrogated by supplementation with exogenous AAT and is likely the result of cellular dysfunction induced by intracellular accumulation of Z-AAT. Using pharmacologic inhibitors, we show that several classes of proteases are involved in matrix degradation by Z-Mɸ. Importantly, compared with normal Mɸ, the membrane-bound serine protease, matriptase, is present in Z-Mɸ at higher levels and contributes to their proteolytic activity on ECM. In addition, we identified matrix metalloproteinase (MMP)-14, a membrane-anchored metalloproteinase, as a novel substrate for matriptase, and showed that matriptase regulates the levels of MMP-14 on the cell surface. Thus, high levels of matriptase may contribute to increased ECM degradation by Z-Mɸ, both directly and through MMP-14 activation. In summary, the expression of Z-AAT in Mɸ confers increased proteolytic activity on ECM. This proteolytic activity is not rescued by exogenous AAT supplementation and could thus contribute to augmentation resistance in AAT deficiency-associated emphysema.

Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells.

Coagulation factor replacement therapy for the X-linked bleeding disorder hemophilia is severely complicated by antibody ("inhibitor") formation. We previously found that oral delivery to hemophilic mice of cholera toxin B subunit-coagulation factor fusion proteins expressed in chloroplasts of transgenic plants suppressed inhibitor formation directed against factors VIII and IX and anaphylaxis against factor IX (FIX). This observation and the relatively high concentration of antigen in the chloroplasts prompted us to evaluate the underlying tolerance mechanisms. The combination of oral delivery of bioencapsulated FIX and intravenous replacement therapy induced a complex, interleukin-10 (IL-10)-dependent, antigen-specific systemic immune suppression of pathogenic antibody formation (immunoglobulin [Ig] 1/inhibitors, IgE) in hemophilia B mice. Tolerance induction was also successful in preimmune mice but required prolonged oral delivery once replacement therapy was resumed. Orally delivered antigen, initially targeted to epithelial cells, was taken up by dendritic cells throughout the small intestine and additionally by F4/80(+) cells in the duodenum. Consistent with the immunomodulatory responses, frequencies of tolerogenic CD103(+) and plasmacytoid dendritic cells were increased. Ultimately, latency-associated peptide expressing CD4(+) regulatory T cells (CD4(+)CD25(-)LAP(+) cells with upregulated IL-10 and transforming growth factor-ß (TGF-ß) expression) as well as conventional CD4(+)CD25(+) regulatory T cells systemically suppressed anti-FIX responses.

Superior In vivo Transduction of Human Hepatocytes Using Engineered AAV3 Capsid.

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.

Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor formation and fatal anaphylaxis in hemophilia B mice.

To address complications of pathogenic antibody or life-threatening anaphylactic reactions in protein replacement therapy for patients with hemophilia or other inherited protein deficiencies, we have developed a prophylactic protocol using a murine hemophilia B model. Oral delivery of coagulation factor IX fused with cholera toxin beta-subunit (with or without a furin cleavage site; CTB-FFIX or CTB-FIX), expressed in chloroplasts (up to 3.8% soluble protein or 0.4 mg/g leaf tissue), bioencapsulated in plant cells, effectively blocked formation of inhibitory antibodies (undetectable or up to 100-fold less than controls). Moreover, this treatment eliminated fatal anaphylactic reactions that occurred after four to six exposures to intravenous F.IX. Whereas only 20-25% of control animals survived after six to eight F.IX doses, 90-93% of F.IX-fed mice survived 12 injections without signs of allergy or anaphylaxis. Immunostaining confirmed delivery of F.IX to Peyer's patches in the ileum. Within 2-5 h, feeding of CTB-FFIX additionally resulted in systemic delivery of F.IX antigen. This high-responder strain of hemophilia B mice represents a new animal model to study anaphylactic reactions. The protocol was effective over a range of oral antigen doses (equivalent to 5-80 microg recombinant F.IX/kg), and controlled inhibitor formation and anaphylaxis long-term, up to 7 months (approximately 40% life span of this mouse strain). Oral antigen administration caused a deviant immune response that suppressed formation of IgE and inhibitory antibodies. This cost-effective and efficient approach of antigen delivery to the gut should be applicable to several genetic diseases that are prone to pathogenic antibody responses during treatment.

Induction of antigen-specific tolerance by hepatic AAV immunotherapy regardless of T cell epitope usage or mouse strain background.

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.

Nonredundant roles of IL-10 and TGF-ß in suppression of immune responses to hepatic AAV-factor IX gene transfer.

Hepatic gene transfer using adeno-associated viral (AAV) vectors has been shown to efficiently induce immunological tolerance to a variety of proteins. Regulatory T-cells (Treg) induced by this route suppress humoral and cellular immune responses against the transgene product. In this study, we examined the roles of immune suppressive cytokines interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß) in the development of tolerance to human coagulation factor IX (hF.IX). Interestingly, IL-10 deficient C57BL/6 mice receiving gene transfer remained tolerant to hF.IX and generated Treg that suppressed anti-hF.IX formation. Effects of TGF-ß blockade were also minor in this strain. In contrast, in C3H/HeJ mice, a strain known to have stronger T-cell responses against hF.IX, IL-10 was specifically required for the suppression of CD8(+) T-cell infiltration of the liver. Furthermore, TGF-ß was critical for tipping the balance toward an regulatory immune response. TGF-ß was required for CD4(+)CD25(+)FoxP3(+) Treg induction, which was necessary for suppression of effector CD4(+) and CD8(+) T-cell responses as well as antibody formation. These results demonstrate the crucial, nonredundant roles of IL-10 and TGF-ß in prevention of immune responses against AAV-F.IX-transduced hepatocytes.

Coaxing the liver into preventing autoimmune disease in the brain.

The liver has several unique immunological properties that affect T cell activation and immune regulation. Recent studies have uncovered opportunities for the treatment of genetic disease by directing expression of the functional therapeutic protein to hepatocytes. In a new study in this issue of the JCI, Lüth and colleagues demonstrate that hepatic expression of a brain protein is protective against neuroinflammatory disease in a mouse model of human MS (see the related article beginning on page 3403). Suppression of autoimmunity was dependent on transgene expression in the liver and was mediated by induction of antigen-specific CD4+CD25+Foxp3+ Tregs. These findings suggest that the introduction of antigens to the liver may have potential as a preventative or therapeutic intervention for autoimmune disease.

High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines.

Elimination of specific surface-exposed single tyrosine (Y) residues substantially improves hepatic gene transfer with adeno-associated virus type 2 (AAV2) vectors. Here, combinations of mutations in the seven potentially relevant Y residues were evaluated for further augmentation of transduction efficiency. These mutant capsids packaged viral genomes to similar titers and retained infectivity. A triple-mutant (Y444+500+730F) vector consistently had the highest level of in vivo gene transfer to murine hepatocytes, approximately threefold more efficient than the best single-mutants, and ~30-80-fold higher compared with the wild-type (WT) AAV2 capsids. Improvement of gene transfer was similar for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors, indicating that these effects are independent of viral second-strand DNA synthesis. Furthermore, Y730F and triple-mutant vectors provided a long-term therapeutic and tolerogenic expression of human factor IX (hF.IX) in hemophilia B (HB) mice after administration of a vector dose that only results in subtherapeutic and transient expression with WT AAV2 encapsidated vectors. In summary, introduction of multiple tyrosine-mutations into the AAV2 capsid results in vectors that yield at least 30-fold improvement of transgene expression, thereby lowering the required therapeutic dose and potentially vector-related immunogenicity. Such vectors should be attractive for treatment of hemophilia and other genetic diseases.

Impact of the underlying mutation and the route of vector administration on immune responses to factor IX in gene therapy for hemophilia B.

Immune responses to factor IX (F.IX), a major concern in gene therapy for hemophilia, were analyzed for adeno-associated viral (AAV-2) gene transfer to skeletal muscle and liver as a function of the F9 underlying mutation. Vectors identical to those recently used in clinical trials were administered to four lines of hemophilia B mice on a defined genetic background [C3H/HeJ with deletion of endogenous F9 and transgenic for a range of nonfunctional human F.IX (hF.IX) variants]. The strength of the immune response to AAV-encoded F.IX inversely correlated with the degree of conservation of endogenous coding information and levels of endogenous antigen. Null mutation animals developed T- and B-cell responses in both protocols. However, inhibitor titers were considerably higher upon muscle gene transfer (or protein therapy). Transduced muscles of Null mice had strong infiltrates with CD8+ cells, which were much more limited in the liver and not seen for the other mutations. Sustained expression was achieved with liver transduction in mice with crm(-) nonsense and missense mutations, although they still formed antibodies upon muscle gene transfer. Therefore, endogenous expression prevented T-cell responses more effectively than antibody formation, and immune responses varied substantially depending on the protocol and the underlying mutation.

Enhanced Transduction of Human Hematopoietic Stem Cells by AAV6 Vectors: Implications in Gene Therapy and Genome Editing.

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 ß-thalassemia and sickle cell disease.

Hepatic gene transfer as a means of tolerance induction to transgene products.

The liver is a preferred target organ for gene therapy not only for liver-specific diseases but also for disorders that require systemic delivery of a protein. Diseases that could benefit from hepatic gene transfer include hemophilia, metabolic disorders, lysosomal storage disorders, and others. For a successful delivery of the transgene and sustained expression, the protocol must avoid immune responses in order to be efficacious. A growing number of studies have demonstrated that liver-directed transfer can induce transgene product-specific immune tolerance. Tolerance obtained via this route requires optimal engineering of the vector to eliminate transgene expression in antigen presenting cells while restricting high levels of therapeutic expression to hepatocytes. Innate immune responses may prevent tolerance induction, cause toxicity, and have to be minimized. Discussed in our review is the crucial role of CD4(+)CD25(+) regulatory T cells in tolerance to the hepatocyte-derived gene product, the immunobiology of the liver and our current understanding of its tolerogenic properties, current and proposed research as to the mechanisms behind the liver's unique cellular environment, as well as development of the tools for tolerance induction such as advanced vector systems.