Ashley T. Martino

The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9-dependent innate immune responses in the liver.

Although adeno-associated viral (AAV) vectors have been successfully used in hepatic gene transfer for treatment of hemophilia and other diseases in animals, adaptive immune responses blocked long-term transgene expression in patients on administration of single-stranded AAV serotype-2 vector. More efficient vectors have been developed using alternate capsids and self-complimentary (sc) genomes. This study investigated their effects on the innate immune profile on hepatic gene transfer to mice. A mild and transient up-regulation of myeloid differentiation primary response gene (88), TLR9, TNF-α, monocyte chemotactic protein-1, IFN-γ inducible protein-10, and IFN-α/β expression in the liver was found after single-stranded AAV vector administration, regardless of the capsid sequence. In contrast, scAAV vectors induced higher increases of these transcripts, upregulated additional proinflammatory genes, and increased circulating IL-6. Neutrophil, macrophage, and natural killer cell liver infiltrates were substantially higher on injection of scAAV. Some but not all of these responses were Kupffer cell dependent. Independent of the capsid or expression cassette, scAAV vectors induced dose-dependent innate responses by signaling through TLR9. Increased innate responses to scAAV correlated with stronger adaptive immune responses against capsid (but not against the transgene product). However, these could be blunted by transient inhibition of TLR9.

Activation of the NF-κB pathway by adeno-associated virus (AAV) vectors and its implications in immune response and gene therapy

Because our in silico analysis with a human transcription factor database demonstrated the presence of several binding sites for NF-κB, a central regulator of cellular immune and inflammatory responses, in the adeno-associated virus (AAV) genome, we investigated whether AAV uses NF-κB during its life cycle. We used small molecule modulators of NF-κB in HeLa cells transduced with recombinant AAV vectors. VP16, an NF-κB activator, augmented AAV vector-mediated transgene expression up to 25-fold. Of the two NF-κB inhibitors, Bay11, which blocks both the canonical and the alternative NF-κB pathways, totally ablated transgene expression, whereas pyrrolidone dithiocarbamate, which interferes with the classical NF-κB pathway, had no effect. Western blot analyses confirmed the abundance of the nuclear p52 protein component of the alternative NF-κB pathway in the presence of VP16, which was ablated by Bay11, suggesting that AAV transduction activates the alternative NF-κB pathway. In vivo, hepatic AAV gene transfer activated the canonical NF-κB pathway within 2 h, resulting in expression of proinflammatory cytokines and chemokines (likely reflecting the sensing of viral particles by antigen-presenting cells), whereas the alternative pathway was activated by 9 h. Bay11 effectively blocked activation of both pathways without interfering with long-term transgene expression while eliminating proinflammatory cytokine expression. These studies suggest that transient immunosuppression with NF-κB inhibitors before transduction with AAV vectors should lead to a dampened immune response, which has significant implications in the optimal use of AAV vectors in human gene therapy.

Innate Immune Responses to AAV Vectors

Gene replacement therapy by in vivo delivery of adeno-associated virus (AAV) is attractive as a potential treatment for a variety of genetic disorders. However, while AAV has been used successfully in many models, other experiments in clinical trials and in animal models have been hampered by undesired responses from the immune system. Recent studies of AAV immunology have focused on the elimination of transgene-expressing cells by the adaptive immune system, yet the innate immune system also has a critical role, both in the initial response to the vector and in prompting a deleterious adaptive immune response. Responses to AAV vectors are primarily mediated by the TLR9–MyD88 pathway, which induces the production of pro-inflammatory cytokines by activating the NF-κB pathways and inducing type I IFN production; self-complementary AAV vectors enhance these inflammatory processes. Additionally, the alternative NF-κB pathway influences transgene expression in cells transduced by AAV. This review highlights these recent discoveries regarding innate immune responses to AAV and discusses strategies to ablate these potentially detrimental signaling pathways.

Tolerance Induction to Cytoplasmic β-Galactosidase by Hepatic AAV Gene Transfer — Implications for Antigen Presentation and Immunotoxicity

Background Hepatic gene transfer, in particular using adeno-associated viral (AAV) vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein. Major Findings AAV-2 vector-mediated hepatic gene transfer for expression of cytoplasmic β-galactosidase (β-gal) was performed in immune competent mice, followed by a secondary β-gal gene transfer with E1/E3-deleted adenoviral Ad-LacZ vector to provoke a severe immunotoxic response. Transgene expression from the AAV-2 vector in ∼2% of hepatocytes almost completely protected from inflammatory T cell responses against β-gal, eliminated antibody formation, and significantly reduced adenovirus-induced hepatotoxicity. Consequently, ∼10% of hepatocytes continued to express β-gal 45 days after secondary Ad-LacZ gene transfer, a time point when control mice had lost all Ad-LacZ derived expression. Suppression of inflammatory T cell infiltration in the liver and liver damage was linked to specific transgene expression and was not seen for secondary gene transfer with Ad-GFP. A combination of adoptive transfer studies and flow cytometric analyses demonstrated induction of Treg that actively suppressed CD8+ T cell responses to β-gal and that was amplified in liver and spleen upon secondary Ad-LacZ gene transfer. Conclusions These data demonstrate that tolerance induction by hepatic AAV gene transfer does not require systemic delivery of the transgene product and that expression of a cytoplasmic neo-antigen in few hepatocytes can induce Treg and provide long-term suppression of inflammatory responses and immunotoxicity.

Nonredundant Roles of IL-10 and TGF-β 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-β (TGF-β) 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-β 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-β was critical for tipping the balance toward an regulatory immune response. TGF-β 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-β in prevention of immune responses against AAV-F.IX-transduced hepatocytes.

Partial correction of the CFTR-dependent ABPA mouse model with recombinant adeno-associated virus gene transfer of truncated CFTR gene

Recently, we have developed a model of airway inflammation in a CFTR knockout mouse utilizing Aspergillus fumigatus crude protein extract (Af‐cpe) to mimic allergic bronchopulmonary aspergillosis (ABPA) 1 , an unusual IgE‐mediated hypersensitivity syndrome seen in up to 15% of cystic fibrosis (CF) patients and rarely elsewhere. We hypothesized that replacement of CFTR via targeted gene delivery to airway epithelium would correct aberrant epithelial cytokine signaling and ameliorate the ABPA phenotype in CFTR‐deficient (CFTR 489X − /−, FABP‐hCFTR + / +) mice. CFTR knockout mice underwent intra‐tracheal (IT) delivery of recombinant adeno‐associated virus serotype 5 (rAAV5Δ‐264CFTR) or rAAV5‐GFP at 2.58 × 1012 viral genomes/mouse. All mice were then sensitized with two serial injections (200 µg) of crude Af antigen via the intra‐peritoneal (IP) route. Untreated mice were sensitized without virus exposure. Challenges were performed 2 weeks after final sensitization, using a 0.25% solution containing Aspergillus fumigatus crude protein extract delivered by inhalation on three consecutive days. The rAAV5Δ‐264CFTR‐treated mice had lower total serum IgE levels (172513 ng/ml ± 1312) than rAAV5‐GFP controls (26 892 ng/ml ± 3715) (p = 0.037) and non‐treated, sensitized controls (24 816 ± 4219 ng/ml). Serum IgG1 levels also were lower in mice receiving the CFTR vector. Interestingly, splenocytes from rAAV5Δ‐264CFTR‐treated mice secreted less IL‐13, INFg, TNFa, RANTES and GM‐CSF after ConA stimulation. Gene therapy with rAAV5Δ‐264CFTR attenuated the hyper‐IgE response in this reproducible CF mouse model of ABPA, with systemic effects also evident in the cytokine response of stimulated splenocytes. Copyright

The pros and cons of immunomodulatory IL-10 gene therapy with recombinant AAV in a Cftr −/− -dependent allergy mouse model

Cystic fibrosis (CF) patients have decreased levels of lung epithelial interleukin (IL)-10 and increased levels of proinflammatory cytokines (tumor necrosis factor-α, IL-4, IL-8 and IL-6). This has also been documented in Cftr (cystic fibrosis transmembrane conductance regulator)-deficient mice (Cftr 489X−/−, FABP-hCFTR+/+). Our laboratory has recently characterized a peculiar hyper-IgE phenotype in these mice, in response to Aspergillus fumigatus crude protein extract (Af-cpe). Thus, we hypothesized that sustained systemic circulating IL-10 levels achieved through skeletal muscle transduction with recombinant adeno-associated vectors expressing IL-10 (rAAV1-IL-10) would serve to downregulate Th1 and Th2 cytokine production. This in turn would dampen the allergic response in the Cftr−/−-dependent mouse model of allergic bronchopulmonary aspergillosis. After Af-cpe sensitization and airway challenge, mice treated with rAAV1-IL-10 had markedly lower IgE levels when compared to the control-treated rAAV1-GFP group. This was accompanied by a significant reduction in the levels of IL-5, IL-4 and IL-13 in the lung compartment. The lower lung cytokine profiles resulted in a near absence of eosinophil recruitment in the lung and a lower inflammatory response in the lung tissue of mice receiving rAAV1-IL-10. Unfortunately, sustained secretion of IL-10 from transduced muscle did lead to thrombocytopenia and splenomegaly in mice injected with rAAV1-IL-10. These results highlight that while IL-10 gene therapy is very effective for treating allergic responses caution must be taken with the prolonged secretion of IL-10.

Pre-Clinical Assessment of Immune Responses to Adeno-Associated Virus (AAV) Vectors

Transitioning to human trials from pre-clinical models resulted in the emergence of inhibitory AAV vector immune responses which has become a hurdle for sustained correction. Early animal studies did not predict the full range of host immunity to the AAV vector in human studies. While pre-existing antibody titers against AAV vectors has been a lingering concern, cytotoxic T-cell (CTL) responses against the input capsid can prevent long-term therapy in humans. These discoveries spawned more thorough profiling of immune response to rAAV in pre-clinical models, which have assessed both innate and adaptive immunity and explored methods for bypassing these responses. Many efforts toward measuring innate immunity have utilized Toll-like receptor deficient models and have focused on differential responses to viral capsid and genome. From adaptive studies, it is clear that humoral responses are relevant for initial vector transduction efficiency while cellular responses impact long-term outcomes of gene transfer. Measuring humoral responses to AAV vectors has utilized in vitro neutralizing antibody assays and transfer of seropositive serum to immunodeficient mice. Overcoming antibodies using CD20 inhibitors, plasmapheresis, altering route of delivery and using different capsids have been explored. CTL responses were measured using in vitro and in vivo models. In in vitro assays expansion of antigen-specific T-cells as well as cytotoxicity toward AAV transduced cells can be shown. Many groups have successfully mimicked antigen-specific T-cell proliferation, but actual transgene level reduction and parameters of cytotoxicity toward transduced target cells have only been shown in one model. The model utilized adoptive transfer of capsid-specific in vitro expanded T-cells isolated from immunized mice with LPS as an adjuvant. Finally, the development of immune tolerance to AAV vectors by enriching regulatory T-cells as well as modulating the response pharmacologically has also been explored.

Role of the vector genome and underlying factor IX mutation in immune responses to AAV gene therapy for hemophilia B

BackgroundSelf-complementary adeno-associated virus (scAAV) vectors have become a desirable vector for therapeutic gene transfer due to their ability to produce greater levels of transgene than single-stranded AAV (ssAAV). However, recent reports have suggested that scAAV vectors are more immunogenic than ssAAV. In this study, we investigated the effects of a self-complementary genome during gene therapy with a therapeutic protein, human factor IX (hF.IX).MethodsHemophilia B mice were injected intramuscularly with ss or scAAV1 vectors expressing hF.IX. The outcome of gene transfer was assessed, including transgene expression as well as antibody and CD8+ T cell responses to hF.IX.ResultsSelf-complementary AAV1 vectors induced similar antibody responses (which eliminated systemic hF.IX expression) but stronger CD8+ T cell responses to hF.IX relative to ssAAV1 in mice with F9 gene deletion. As a result, hF.IX-expressing muscle fibers were effectively eliminated in scAAV-treated mice. In contrast, mice with F9 nonsense mutation (late stop codon) lacked antibody or T cell responses, thus showing long-term expression regardless of the vector genome.ConclusionsThe nature of the AAV genome can impact the CD8+ T cell response to the therapeutic transgene product. In mice with endogenous hF.IX expression, however, this enhanced immunogenicity did not break tolerance to hF.IX, suggesting that the underlying mutation is a more important risk factor for transgene-specific immunity than the molecular form of the AAV genome.

Interleukin 10 (IL-10) Regulatory Cytokine and its Clinical Consequences

IL-10 involvement in disease progression continues to be evaluated. Through these studies it has become evident that the role of IL-10 in immunological conditions encompasses a large range of disorders. IL-10 deficiencies can lead to Th1 hypersensitivities i.e. Celiac’s disease and autoimmue disorders i.e. type 1 diabetes (TID). Conversely, increased IL-10 results in Th2 related hypersensitivities i.e. allergic dermatitis and autoimmune disorders i.e. systemic lupus erythematosus (SLE). These polar conditions are related to increases in either Th1 cytokines or Th2 cytokines respectively. With the dominant role of IL-10 being regulatory, clinical consequences can result from IL-10 related immuno-suppression. These enhanced IL-10 regulatory responses are related to improper clearance of pathogens and tumor cells, resulting in chronic infections and tumor development. Interestingly, HPV, HCV, HBV and other common chronic pathogens can persist even with normal levels of IL-10 but can be cleared by inhibiting IL-10 function.