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.
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.
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.