Herzog Lab

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.

Herzog RW, Kaczmarek R, High KA (2025) Gene therapy for hemophilia – from basic science to first approvals of “one-and-done” therapies. Mol Ther 33: 2015-2034

Kumar SRP, Biswas M, Cao D, Arisa S, Muñoz-Melero M, Lam AK, Piñeros AR, Kapur R, Kaisho T, Kaufman RJ, Xiao W, Shayakhmetov DM, Terhorst C, de Jong YP, Herzog RW (2024) TLR9-independent CD8+ T cell responses in hepatic AAV gene transfer through IL-1R1-MyD88 signaling. Mol Ther 32: 325-339 

Kaczmarek R, Piñeros AR, Patterson PE, Bertolini TB, Perrin GQ, Sherman A, Born J, Arisa S, Arvin MC, Kamocka MM, Martinez MM, Dunn KW, Quinn SQ, Morris JJ, Wilhelm AR, Kaisho T, Muñoz-Melero M, Biswas M, Kaplan MH, Linnemann AK, George LA, Camire RM, Herzog RW (2023) Factor VIII trafficking to CD4+ T cells shapes its immunogenicity and requires several types of antigen presenting cells. Blood 142: 290-305

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 30: 3552-3569

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

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