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Browsing by Author "Zolotukhin, Irene"
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Item Effect of CpG Depletion of Vector Genome on CD8+ T Cell Responses in AAV Gene Therapy(Frontiers Media, 2021-05-31) Bertolini, Thais B.; Shirley, Jamie L.; Zolotukhin, Irene; Li, Xin; Kaisho, Tsuneyasu; Xiao, Weidong; Kumar, Sandeep R.P.; Herzog, Roland W.; Pediatrics, School of MedicineAdeno associated viral (AAV) vectors have emerged as a preferred platform for in vivo gene replacement therapy and represent one of the most promising strategies to treat monogenetic disorders such as hemophilia. However, immune responses to gene transfer have hampered human gene therapy in clinical trials. Over the past decade, it has become clear that innate immune recognition provides signals for the induction of antigen-specific responses against vector or transgene product. In particular, TLR9 recognition of the vector's DNA genome in plasmacytoid dendritic cells (pDCs) has been identified as a key factor. Data from clinical trials and pre-clinical studies implement CpG motifs in the vector genome as drivers of immune responses, especially of CD8+ T cell activation. Here, we demonstrate that cross-priming of AAV capsid-specific CD8+ T cells depends on XCR1+ dendritic cells (which are likely the main cross-presenting cell that cooperates with pDCs to activate CD8+ T cells) and can be minimized by the elimination of CpG motifs in the vector genome. Further, a CpG-depleted vector expressing human coagulation factor IX showed markedly reduced (albeit not entirely eliminated) CD8+ T cell infiltration upon intramuscular gene transfer in hemophilia B mice when compared to conventional CpG+ vector (comprised of native sequences), resulting in better preservation of transduced muscle fibers. Therefore, this deimmunization strategy is helpful in reducing the potential for CD8+ T cell responses to capsid or transgene product. However, CpG depletion had minimal effects on antibody responses against capsid or transgene product, which appear to be largely independent of CpG motifs.Item Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity(Elsevier, 2020-10) Biswas, Moanaro; Marsic, Damien; Li, Ning; Zou, Chenhui; Gonzalez-Aseguinolaza, Gloria; Zolotukhin, Irene; Kumar, Sandeep R.P.; Rana, Jyoti; Butterfield, John S.S.; Kondratov, Oleksandr; de Jong, Ype P.; Herzog, Roland W.; Zolotukhin, Sergei; Pediatrics, School of MedicineLimitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. In vivo, AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies.Item Type I IFN Sensing by cDCs and CD4+ T Cell Help Are Both Requisite for Cross-Priming of AAV Capsid-Specific CD8+ T Cells(Elsevier, 2019) Shirley, Jamie L.; Keeler, Geoffrey D.; Sherman, Alexandra; Zolotukhin, Irene; Markusic, David M.; Hoffman, Brad E.; Morel, Laurence M.; Wallet, Mark A.; Terhorst, Cox; Herzog, Roland W.; Pediatrics, School of MedicineAdeno-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.