The research laboratory of Roland Herzog, PhD, develops adeno-associated viral (AAV) based gene therapies and immune tolerance induction protocols for the X-linked bleeding disorder hemophilia and other diseases. Investigations aim at uncovering immune mechanisms leading to rejection or, conversely, lasting therapy. Detailed studies address the interactions between the viral vector and its transgene products and the immune system.
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Innate and adaptive immunity to AAV vectors
Successful gene therapy requires that neither the gene transfer vector nor the therapeutic gene product are being rejected by the immune system. Initial immune recognition occurs through the innate immune system, which then provides activation signals to the adaptive immune system, potentially resulting in cytotoxic T lymphocyte responses and antibody formation. Adeno-associated viral (AAV) vectors provoke little innate immunity compared to other vectors or viruses, making it a preferable vector for in vivo gene therapy. However, innate immune sensing of AAV does occur and is consequential. We previously found that sensing of AAV’s DNA genome by TLR9 in plasmacytoid dendritic cells (pDCs) serves as an activation signal for priming of CD8+ T cell (e.g. cross-priming of capsid-specific CD8+ T cells), which involves co-operation of pDCs with cross-presenting conventional DCs (cDCs) expressing the receptor for IFN I. The Herzog lab is now identifying other innate immune sensing and cytokine signaling pathways that link innate immune recognition of viral gene transfer to antigen-specific responses in different organs. Furthermore, the lab is testing various vector engineering and immune modulatory approaches to prevent unwanted immune responses.
Immune tolerance induction by hepatic gene transfer
Interestingly, gene transfer to hepatocytes induces FoxP3+ regulatory T cells (Treg) and may lead to induction of immune tolerance to the transgene product. Therefore, liver gene transfer can be used both for treatment of disease and for immunotherapy. The Gene and Cell Therapy Program continues to study the mechanism of tolerance induction in the hepatic environment and utilizes the approach to achieve long-term correction of genetic disease and prevention or reversal of acquired immune diseases such as allergies. Organ-specific immune responses to Cas9 are also being investigated in the context of in vivo gene editing.
The challenge of factor VIII gene transfer
Gene therapy for hemophilia A (factor VIII deficiency) is advancing clinically but faces multiple complications, including liver toxicity and lack of durability. These features are likely related to unique complications of factor VIII expression. This protein that is prone to misfolding and very immunogenic. Through collaboration with multiple laboratories at IU and other institutions, the Herzog lab is addressing basic questions about the immunogenicity and biology of factor VIII expression in hepatocytes. These include contributions of innate and adaptive immunity, mechanisms of antigen presentation, ER stress, mechanisms of shutdown of gene expression, and, importantly, avenues to achieve sustained therapy such as factor VIII variants that are better secreted, immune modulation, and interventions that lower cellular stress.
Oral tolerance induction
Introduction of antigens to the immune system of the small intestine represents an alternative tolerogenic route. Prior work by the Herzog lab has shown that repeated oral delivery of transgenic plant cells expressing factor VIII or factor IX antigens fused to a transmucosal carrier protein induces LAP+ and FoxP3+ (and dual positive) Treg that suppress antibody formation in subsequent intravenous replacement therapy for hemophilia. Oral delivery of immune modulatory antibodies such as anti-CD3 also has the potential for suppressing anti-drug antibody formation in protein replacement therapy. Ongoing work compares and contrasts efficacy and mechanisms of these approaches, with particular emphasis on phenotypes and characteristics of Treg subsets.
Current Research Funding
Toward safer gene therapy for hemophilia A
Enhancing immune regulation in gene therapy for hemophilia
Affordable oral delivery of human blood protein drugs bioencapsulated in plant cells.
Molecular and cellular mechanisms of the Factor VIII immune response
Immunology of factor IX gene transfer to liver
Butterfield JSS, Yamada K, Bertolini TB, Syed F, Kumar SRP, Li X, Arisa S, Piñeros AR, Tapia A, Rogers CA, Li N, Rana J, Biswas M, Terhorst C, Kaufman RJ, de Jong YP, Herzog RW (2022) IL-15 blockade and rapamycin rescue multifactorial loss of factor VIII from AAV-transduced hepatocytes in hemophilia A mice. Mol Ther, online ahead of print (18 pages)
Arruda VR, Lillicrap D, and Herzog RW (2022) Immune complications and their management in inherited and acquired bleeding disorders. Blood 140: 1075-1085
Hakim CH, Kumar SRP, Perez-Lopez DO, Wasala NB, Zhang D, Yue Y, Teixeira J, Pan X, Zhang K, Million ED, Nelson CE, Metzger S, Han J, Louderman JA, Schmidt F, Feng F, Grimm D, Smith, BF Yao G, Yang NN, Gersbach CA, Chen S, Herzog RW, Duan D (2021) Local and systemic AAV CRISPR therapy induces Cas9-specific immune responses in the canine model of Duchenne muscular dystrophy. Nat. Commun. 12: 6769
Shirley JL, de Yong YP, Terhorst C, Herzog RW (2020) Immune responses to viral gene therapy vectors. Mol Ther 28: 709-722
Shirley JL, Keeler GD, Sherman A, Zolotukhin I, Markusic DM, Hoffman BE, Morel LM, Wallet MA, Terhorst C, Herzog RW (2020) Type I IFN sensing by cDCs and CD4+ T Cell help are both requisite for cross-priming of AAV capsid-specific CD8+ T cells. Mol. Ther. 28: 758-770
Sandeep Kumar, PhD, Assistant Research Professor
Radoslaw Kaczmarek, PhD, postdoctoral fellow
Sreevani Arisa, BS, research analyst
Kentaro Yamada, PhD, assistant scientist
Miguel Gonzales, PhD, postdoctoral fellow
Paige Patterson, MD, pediatric hematology/oncology fellow
John S. Butterfield, BS, graduate student (MD/PhD program)
Xin Li, MS, research associate
Maite Munoz, BS, research technician