Geoffrey L. Rogers

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.

Differential Type I Interferon-dependent Transgene Silencing of Helper-dependent Adenoviral vs. Adeno-associated Viral Vectors In Vivo

We previously dissected the components of the innate immune response to Helper-dependent adenoviral vectors (HDAds) using genetic models, and demonstrated that multiple pattern recognition receptor signaling pathways contribute to this host response to HDAds in vivo. Based on analysis of cytokine expression profiles, type I interferon (IFN) mRNA is induced in host mouse livers at 1 hour post-injection. This type I IFN signaling amplifies cytokine expression in liver independent of the nature of vector DNA sequences after 3 hours post-injection. This type I IFN signaling in response to HDAds administration contributes to transcriptional silencing of both HDAd prokaryotic and eukaryotic DNA in liver. This silencing occurs early and is mediated by epigenetic modification as shown by in vivo chromatin immunoprecipitation (ChIP) with anti-histone deacetylase (HDAC) and promyelocytic leukemia protein (PML). In contrast, self-complementary adeno-associated viral vectors (scAAVs) showed significantly lower induction of type I IFN mRNA in liver compared to HDAds at both early and late time points. These results show that the type I IFN signaling dependent transgene silencing differs between AAV and HDAd vectors after liver-directed gene transfer.

Gene therapy for hemophilia

Hemophilia is an X-linked inherited bleeding disorder consisting of two classifications, hemophilia A and hemophilia B, depending on the underlying mutation. Although the disease is currently treatable with intravenous delivery of replacement recombinant clotting factor, this approach represents a significant cost both monetarily and in terms of quality of life. Gene therapy is an attractive alternative approach to the treatment of hemophilia that would ideally provide life-long correction of clotting activity with a single injection. In this review, we will discuss the multitude of approaches that have been explored for the treatment of both hemophilia A and B, including both in vivo and ex vivo approaches with viral and nonviral delivery vectors.

Synergy between rapamycin and FLT3 ligand enhances plasmacytoid dendritic cell–dependent induction of CD4+CD25+FoxP3+ Treg

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Treg) are critical elements for maintaining immune tolerance, for instance to exogenous antigens that are introduced during therapeutic interventions such as cell/organ transplant or gene/protein replacement therapy. Coadministration of antigen with rapamycin simultaneously promotes deletion of conventional CD4(+) T cells and induction of Treg. Here, we report that the cytokine FMS-like receptor tyrosine kinase ligand (Flt3L) enhances the in vivo effect of rapamycin. This occurs via selective expansion of plasmacytoid dendritic cells (pDCs), which further augments the number of Treg. Whereas in conventional DCs, rapamycin effectively blocks mammalian target of rapamycin (mTOR) 1 signaling induced by Flt3L, increased mTOR1 activity renders pDCs more resistant to inhibition by rapamycin. Consequently, Flt3L and rapamycin synergistically promote induction of antigen-specific Treg via selective expansion of pDCs. This concept is supported by the finding that Treg induction is abrogated upon pDC depletion. The combination with pDCs and rapamycin is requisite for Flt3L/antigen-induced Treg induction because Flt3L/antigen by itself fails to induce Treg. As co-administering Flt3L, rapamycin, and antigen blocked CD8(+) T-cell and antibody responses in models of gene and protein therapy, we conclude that the differential effect of rapamycin on DC subsets can be exploited for improved tolerance induction.

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.

Unique Roles of TLR9- and MyD88-Dependent and -Independent Pathways in Adaptive Immune Responses to AAV-Mediated Gene Transfer

The immune system represents a significant barrier to successful gene therapy with adeno-associated viral (AAV) vectors. In particular, adaptive immune responses to the viral capsid or the transgene product are of concern. The sensing of AAV by toll-like receptors (TLRs) TLR2 and TLR9 has been suggested to play a role in innate immunity to the virus and may also shape subsequent adaptive immune responses. Here, we investigated the functions of TLR2, TLR9 and the downstream signaling adaptor MyD88 in antibody and CD8+ T-cell responses. Antibody formation against the transgene product occurred largely independently of TLR signaling following gene transfer with AAV1 or AAV2 vectors, whereas loss of signaling through the TLR9-MyD88 pathway substantially reduced CD8+ T-cell responses. In contrast, MyD88 (but neither of the TLRs) regulated antibody responses to capsid. B cell-intrinsic MyD88 was required for the formation of anti-capsid IgG2c independently of vector serotype or route of administration. However, MyD88-/- mice instead produced anti-capsid IgG1 that emerged with delayed kinetics but nonetheless completely prevented in vivo readministration. We conclude that there are distinct roles for TLR9 and MyD88 in promoting adaptive immune responses to AAV-mediated gene transfer and that there are redundant MyD88-dependent and MyD88-independent mechanisms that stimulate neutralizing antibody formation against AAV.

Optimal Immunofluorescent Staining for Human Factor IX and Infiltrating T Cells following Gene Therapy for Hemophilia B

Immunofluorescent imaging is a valuable tool for investigating the outcome of gene therapy within the transduced tissue. With a multi-labeling technique, it is possible to both characterize local expression of the transgene and to evaluate the severity of the adaptive immune response through cytotoxic T cell infiltration. It is critical that the experimental parameters are optimal in order to prevent misinterpretation of important pathological events. To optimize this staining protocol, murine liver and skeletal muscle was transduced using recombinant adeno-associated virus encoding human factor IX. After testing several common cryo-preservative and fixative techniques, we found that optimal tissue integrity and antigen (factor IX and CD8) detection was achieved by freezing muscle tissue on liquid nitrogen-cooled isopentane (also called methylbutane or 2-methylbutane), followed by fixation with acetone at room temperature. The staining protocol described herein requires only about two hours, yet maintains exquisite specificity even at high magnification under confocal microscopy.

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.

Immune responses to human factor IX in haemophilia B mice of different genetic backgrounds are distinct and modified by TLR4.

Our laboratory develops protocols to prevent or reverse ongoing anti‐hFIX IgG inhibitors in haemophilia B mice with a F9 gene deletion on BALB/c and C3H/HeJ backgrounds. C3H/HeJ F9−/Y mice develop high titre anti‐hFIX IgG1 inhibitors and anaphylaxis, whereas most BALB/c F9−/Y mice have mild anti‐hFIX IgG1 inhibitors and no anaphylaxis. Our aim was to determine if hFIX‐specific B‐ and T‐cell responses in BALB/c and C3H/HeJ F9−/Y mice trigger the difference in anti‐hFIX immune responses. BALB/c and C3H/HeJ F9−/Y mice were challenged weekly with recombinant hFIX protein. Humoral immune responses were determined by IgG1 and IgG2a anti‐hFIX ELISA, Bethesda assay for inhibitors and B‐cell ELISpot on bone marrow and spleen cells. T‐cell studies measured the TH1 (IFN‐γ) and TH2 (IL‐4) cytokine responses in splenocytes at the mRNA and protein level in response to hFIX protein. Antibody responses were also measured in C3H/HeJ/OuJ F9−/Y mice with restored toll‐like receptor 4 (TLR4) function. BALB/c F9−/Y mice have a TH2 skewed response and a reduction in anti‐hFIX secreting plasma cells in the bone marrow. Independent antigen challenge revealed both strains generated equivalent IgG1 antibody titres to an intravenously delivered antigen. C3H/HeJ F9−/Y mice have a mixed TH1 and TH2 response (mainly TH2). Importantly, TLR4 signalling has a modulatory role in the C3H background on the levels of anti‐hFIX IgG1 and incidence of anaphylaxis. The background strain strongly impacts the immune response to hFIX, which can be significantly impacted by mutations in innate immune sensors.

519. Unique Role of the TLR9-MyD88 Signaling Pathway in Dendritic Cells in AAV Capsid-Specific CD8+ T Cell Activation

Clinical trials of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B have revealed a deleterious role for memory CD8+ T cell responses to the AAV capsid, which can eliminate transduced hepatocytes and transgene expression. However, the mechanisms underlying this response remain unclear, and have been difficult to study because the functional anti-capsid CTL response is not observed in murine models. To better identify capsid-specific T cells, we have created a modified AAV2 vector with a series of substitution mutations that contains the peptide sequence SIINFEKL, which is the CD8+ immunodominant epitope of the model antigen ovalbumin in C57BL/6 mice. With this vector, capsid-specific CD8+ T cells can be identified using an H2-Kb-SIINFEKL tetramer. We have used this vector to study the innate immune mechanisms underlying the formation of AAV capsid-specific CTLs—which pattern recognition receptors and which antigen-presenting cell types are required. Using knockout mice, we determined that sensing of the viral genome by toll-like receptor 9 (TLR9) and its downstream signaling through MyD88 are required for the formation of tetramer-positive (capsid-specific) CD8+ T cells after i.m. injection of AAV2-SIINFEKL. Similarly, OT-1 CD8+ T cells (TCR transgenic specific for SIINFEKL) adoptively transferred to wild-type (WT) mice proliferated following administration of AAV2-SIINFEKL but not with unmodified AAV2 lacking the SIINFEKL motif. In contrast, there was a lack of proliferation in TLR9−/- mice. TLR2 (which has been reported to sense the AAV capsid), AP3 (required for type I interferon signaling downstream of TLR9), and STING (a downstream signaling adaptor of several cytoplasmic sensors of DNA) were dispensable for the anti-capsid CTL response. In terms of the antigen presenting cell requirements, we found that transient depletion of dendritic cells (DCs) in CD11c-DTR mice delayed the development of tetramer-positive CD8+ T cells relative to WT mice. The response in mice that received gadolinium chloride to inactivate macrophages or in B cell-deficient μMT mice was comparable to WT mice. To test the hypothesis that TLR9-MyD88 signaling in DCs is required for capsid-specific CTL responses, we have crossed mice expressing Cre recombinase under the control of the CD11c promoter with mice in which the MyD88 gene is flanked by LoxP sites, creating DC-MyD88−/- mice which specifically lack MyD88 expression—and thus TLR9 signaling—in DCs. Moreover, we have acquired inhibitors of TLR9 and MyD88 to determine if pharmacological inhibition of these pathways is a viable strategy to prevent the formation of capsid-specific CD8+ T cells. In conclusion, TLR9-MyD88 signaling, most likely in DCs, is required for the formation of de novo anti-capsid CD8+ T cell responses.