Timing of Intensive Immunosuppression Impacts Risk of Transgene Antibodies after AAV Gene Therapy


Hemophilia is an X-linked inherited bleeding disorder caused by mutations in the F8 (hemophilia A) or F9 (hemophilia B) genes, resulting in reduced production/function of the factor VIII (FVIII) or factor IX (FIX) proteins. Novel therapeutics to improve care for persons with hemophilia and other genetic diseases that has shown signs of success is the Adeno-associated virus (AAV) vector gene therapy. There are 3 possible immunological obstructions an AAV-based gene therapy has to overcome to be considered successful: (1) vector transduction impediment by already existing anti-AAV neutralizing antibodies, (2) transduced cells eradication by anti-AAV-capsid T cells (cellular immune response), and (3) immune response development against the transgene-product. For each of these obstructions, the intricate relationship between them and immune mechanisms are not fully understood. Some studies have suggested that addressing any one of these limitations by designing immunosuppression may worsen others.

Scientists from the Children’s Hospital of Philadelphia: Dr. Benjamin Samelson-Jones, Jonathan Finn, Dr. Patricia Favaro, Dr. J. Fraser Wright and Dr. Valder Arruda sought the time intensive T cell-directed immunosuppression and measure its impact on rabbit anti-thymocyte globulin (ATG) in non-human primates receiving therapeutically relevant doses of AAV-human factor IX (hFIX) vectors. Their results showed that anti-FIX antibodies were observed with early administration of rabbit ATG accompanied by AAV administration anti-FIX antibodies. The research work is published in the Journal Molecular Therapy. Methods & Clinical Development.

After the administration of ATG, the research team observed a significant decrease in lymphocyte numbers in each group of animals. They also noticed that shortly after ATG administration, there was a transient increase in the frequency of CD4+CD25+FoxP3+ cells in all animals; however, the size of this increase was varied among the animals. ATG was not found to have any depleting effects on platelets and red cell and platelet counts.

The early administration of ATG accompanied by AAV administration was found to be associated with the development of antibodies against the hFIX transgene-product. However, when the administration of ATG was delayed, there was no detectable immune response against hFIX transgene-product, either T cell mediated or humoral. The formation of an inhibitor to hFIX was found to be associated with a Th17 cytokine signature and an increase in the frequency of Th17 cells.

The administration of ATG was well tolerated in their preclinical studies and did not results in an increase the risk for opportunistic infections or have any effect on body weight. In addition, there was no increase in T cell secretion of IFN-γ to AAV2 capsid proteins or hFIX neither was any humoral responses to the transgene product. The only abnormality that was observed a spike in the MMF levels.

An examination of the anti-AAV2 IgG antibodies titer showed that the immunosuppression regimens did not stop humoral responses to the vector capsid. The early administration of ATG seemed to partially inhibit the onset of the humoral anti-capsid response, resulting in a rapid increase in antibody titers following immunosuppression withdrawal in all three animals

The researchers have been able to establish a specific time period around the administration of vector during which rigorous immunosuppression can alter the immune system toward immunogenicity and away from immune tolerance. The findings of the study will advance the successful development of immunosuppression regimens for AAV-based gene therapy.


Samelson-Jones BJ, Finn JD, Favaro P, Wright JF, Arruda VR. Timing of Intensive Immunosuppression Impacts Risk of Transgene Antibodies after AAV Gene Therapy in Nonhuman Primates. Mol Ther Methods Clin Dev. 2020;17:1129-1138.

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