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Faculty Research Labs

Markusic Lab

The research laboratory of David M. Markusic, PhD, MSC, is focused on the characterization and optimization of adeno-associated virus (AAV) liver gene therapy to prevent immune responses directed against the AAV capsid and the transgene product. This research work has made vital contributions to advancing liver directed AAV gene therapy as a novel approach to eradicate pathogenic antidrug antibodies in hemophilia which was extended to treat autoimmune disease and food allergy.

The Markusic Lab is located within the Herman B Wells Center for Pediatric Research at Indiana University School of Medicine.

Active Research

AAV Immunotherapy depicts different pathways of the immune system that sense AAV vectors and presents known and future targets to blunt the immune response.The long-term goal of the Markusic Lab's research is to develop protocols for induction of antigen specific immune tolerance for translation in gene therapy, autoimmune disease, and allergy. Dr. Markusic developed a novel therapy using immune tolerance from AAV-FIX liver gene transfer to suppress and eliminate pre-existing pathogenic anti-FIX antibodies. Similar studies have also been conducted in large animal models including hemophilia dogs and non-human primates. In cases where hepatic AAV expression failed to eliminate antidrug antibodies the lab explored adjunct immunotherapies. Dr. Markusic co-developed an AAV gene immunotherapy platform to treat a mouse model of the autoimmune disease multiple sclerosis that successfully prevented and reversed severe paralysis in mice when combined with a short course of the immune suppressive drug rapamycin. Finally, the Markusic Lab recently published a paper showing that AAV liver gene therapy could treat food allergy in a mouse model of egg allergy. Dr. Markusic's present research interests are to determine the mechanisms of immune tolerance from a hepatic AAV expressed antigen and to study and prevent immunotoxicities related to systemically administered AAV vectors.
                                         

Scientific graphic showing a graphical abstract from a recently published paper that depicts immune tolerance mechanisms for AAV gene immunotherapy in a mouse model of egg food allergy. The potential for immunological rejection of a therapeutic protein remains unknown in hepatic adeno-associated virus (AAV) gene therapy in humans. Despite this, clinical trials for hepatic AAV gene therapy for hemophilia A (factor VIII deficiency) and B (factor IX deficiency) have advanced into phase III and many phase I/II trials are recruiting for patients with inherited disorders of metabolism. Hemophilia is a coagulation disorder in which patients are currently managed with FVIII or FIX protein replacement therapy. Because patients are adults with long-term exposure to recombinant/plasma derived FVIII and FIX proteins, hepatic AAV clinical trial participants are at low risk for forming anti-drug antibodies, termed inhibitors and rejecting their gene therapy. In contrast to hemophilia, many inherited disorders of metabolism have no enzyme replacement therapies or alternative treatments beyond orthotopic liver translation. Therefore, there is a significant gap in knowledge on the potential risk of immune responses to therapeutic proteins following hepatic AAV gene delivery in both immunologically naïve patients and in patients that are predisposed to or have pre-existing immunity to their therapeutic protein. Pre-clinical studies in mice and large animal models show that hepatic AAV antigen expression induces antigen specific peripheral regulatory T cells (Tregs). However, immune tolerance is not always assured with hepatic AAV gene delivery in both naïve animals and in animals with pre-existing immunity to the expressed antigen. Thus, one of the Markusic Lab's current research interests is to address this critical gap in the understanding of the underlying mechanisms driving peripheral Treg induction and immune tolerance to improve outcomes for hepatic AAV gene therapy. 

a brief overview of our hypothesis for regulatory T cell (Treg) induction in response to hepatic AAV gene therapy.

 Despite the widespread success of AAV vectors in pre-clinical and clinical studies, immunotoxicities, primarily related to the AAV capsid, develop in patients resulting in either a reduction or complete elimination of therapeutic protein expression. Ongoing clinical trials report liver toxicity and complement mediated toxicities following systemic AAV vector delivery. The first reported immunotoxicity related to AAV vectors identified an expansion of capsid reactive CD8 T cells that eliminated AAV transduced hepatocytes in hemophilia B patients. However, preclinical testing failed to predict this outcome. Dr. Markusic co-developed the first mouse model that could replicate the hepatic immunotoxicities observed within patients. However, to advance safer and more effective AAV gene therapies into the clinic, researchers need to better understand what triggers these immunotoxicities in humans and how to modify the vector and treatment protocol to prevent immunological rejection of therapy. 

Research Funding

Herman B Wells Center Bridge Grant

Recent Publications

A full list of publications by David M. Markusic, PhD, MSC, are available on PubMed.
  • 2022
    Gonzalez-Visiedo M, Kulis MD, Markusic DM. Manipulating the microbiome to enhance oral tolerance in food allergy. Cell Immunol. 2022 Dec;382:104633. doi: 10.1016/j.cellimm.2022.104633. Epub 2022 Oct 22. PubMed PMID: 36347161.

    Gonzalez-Visiedo M, Li X, Munoz-Melero M, Kulis MD, Daniell H, Markusic DM. Single-dose AAV vector gene immunotherapy to treat food allergy. Mol Ther Methods Clin Dev. 2022 Sep 8;26:309-322. doi: 10.1016/j.omtm.2022.07.008. eCollection 2022 Sep 8. PubMed PMID: 35990748; PubMed Central PMCID: PMC9361215.
  • 2020
    Biswas M, Palaschak B, Kumar SRP, Rana J, Markusic DM. B Cell Depletion Eliminates FVIII Memory B Cells and Enhances AAV8-coF8 Immune Tolerance Induction When Combined With Rapamycin. Front Immunol. 2020;11:1293. doi: 10.3389/fimmu.2020.01293. eCollection 2020. PubMed PMID: 32670285; PubMed Central PMCID: PMC7327091.
  • 2018
    Keeler GD, Kumar S, Palaschak B, Silverberg EL, Markusic DM, Jones NT, Hoffman BE. Gene Therapy-Induced Antigen-Specific Tregs Inhibit Neuro-inflammation and Reverse Disease in a Mouse Model of Multiple Sclerosis. Mol Ther. 2018 Jan 3;26(1):173-183. doi: 10.1016/j.ymthe.2017.09.001. Epub 2017 Sep 21. PubMed PMID: 28943274; PubMed Central PMCID: PMC5762980.
  • 2017
    Palaschak B, Marsic D, Herzog RW, Zolotukhin S, Markusic DM. An Immune-Competent Murine Model to Study Elimination of AAV-Transduced Hepatocytes by Capsid-Specific CD8+ T Cells. Mol Ther Methods Clin Dev. 2017 Jun 16;5:142-152. doi: 10.1016/j.omtm.2017.04.004. eCollection 2017 Jun 16. PubMed PMID: 28480313; PubMed Central PMCID: PMC5415329.
  • 2016
    Zolotukhin I, Markusic DM, Palaschak B, Hoffman BE, Srikanthan MA, Herzog RW. Potential for cellular stress response to hepatic factor VIII expression from AAV vector. Mol Ther Methods Clin Dev. 2016;3:16063. doi: 10.1038/mtm.2016.63. eCollection 2016. PubMed PMID: 27738644; PubMed Central PMCID: PMC5040172.
  • 2015
    Biswas M, Sarkar D, Kumar SR, Nayak S, Rogers GL, Markusic DM, Liao G, Terhorst C, Herzog RW. Synergy between rapamycin and FLT3 ligand enhances plasmacytoid dendritic cell-dependent induction of CD4+CD25+FoxP3+ Treg. Blood. 2015 May 7;125(19):2937-47. doi: 10.1182/blood-2014-09-599266. Epub 2015 Apr 1. PubMed PMID: 25833958; PubMed Central PMCID: PMC4424416.
  • 2013

    Markusic DM, Hoffman BE, Perrin GQ, Nayak S, Wang X, LoDuca PA, High KA, Herzog RW. Effective gene therapy for haemophilic mice with pathogenic factor IX antibodies. EMBO Mol Med. 2013 Nov;5(11):1698-709. doi: 10.1002/emmm.201302859. Epub 2013 Sep 16. PubMed PMID: 24106230; PubMed Central PMCID: PMC3840486.

    Martino AT, Basner-Tschakarjan E, Markusic DM, Finn JD, Hinderer C, Zhou S, Ostrov DA, Srivastava A, Ertl HC, Terhorst C, High KA, Mingozzi F, Herzog RW. Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells. Blood. 2013 Mar 21;121(12):2224-33. doi: 10.1182/blood-2012-10-460733. Epub 2013 Jan 16. PubMed PMID: 23325831; PubMed Central PMCID: PMC3606062.

  • 2011
    Martino AT, Suzuki M, Markusic DM, Zolotukhin I, Ryals RC, Moghimi B, Ertl HC, Muruve DA, Lee B, Herzog RW. The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9-dependent innate immune responses in the liver. Blood. 2011 Jun 16;117(24):6459-68. doi: 10.1182/blood-2010-10-314518. Epub 2011 Apr 7. PubMed PMID: 21474674; PubMed Central PMCID: PMC3123017.

Faculty Research Team

42000-Markusic, David

David M. Markusic, PhD, MSC

Associate Research Professor of Pediatrics

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Additional Research Team Members

Miguel Gonzalez-Visiedo, Postdoctoral Research Associate