D. T. Lee1,2, C. Woo2, C. Tran2, G. S. Lipshutz2 1Mount Sinai School Of Medicine,Department Of Surgery,New York, NY, USA 2University Of California – Los Angeles,Department Of Surgery,Los Angeles, CA, USA
Introduction: Gene therapy is a promising strategy for treatment of inherited genetic disorders. Its efficacy, however, is limited by a potential immune response against transgene encoded neoantigens and the consequent diminution of therapeutic protein levels. Prior studies have shown that neonatal exposure to recombinant viral vector and transgene proteins can result in immunologic tolerance and persistent transgene expression. In this study we sought to determine if central tolerance plays a role in the mechanism of neonatal tolerance via thymic deletion.
Methods: An adeno-associated viral vector (AAV) was developed that expressed secreted ovalbumin (OVA) and green fluorescent protein (GFP) and was administered to C57BL/6 pups at birth; control mice received no virus. OVA and anti-OVA levels were serially obtained for over 1 year. Vaccination with OVA administered subcutaneously was performed. At 6 weeks and 1 year euthanasia was performed and viral DNA was quantified using qPCR. OT-2 mice that express CD4+ transgenic T cell receptors specific for OVA peptide received either no virus at birth or were administered AAV-OVA. OT-2 mice crossed with transgenic mice expressing membrane bound chicken ovalbumin on all cells (CAG-OVA) were injected with AAV-OVA neonatally. Thymi were removed for flow cytometry analysis as adults.
Results: A single intravenous dose of AAV administered to C57BL/6 pups led to life-long expression of ovalbumin in wild type mice (>1 year) without a humoral immune response; this persisted after vaccination with adjuvant. Quantitative PCR revealed that the highest viral copy number levels after 1 year were expressed in the liver. OT-2 mice that received no injection were found to have 81.3% CD4+ thymocytes when examined as adults, while OT-2 mice crossed with CAG-OVA mice were found to have 10.1% of their thymocytes CD4+. OT-2 mice administered AAV-OVA at birth demonstrated 69.6% of their thymocytes as CD4+ (p=0.01 compared to OT-2).
Conclusion: These studies show that neonatally administered AAV-OVA in C57BL/6 mice results in long term protein expression and tolerance to OVA. After vaccination challenge with a purified OVA protein, serum OVA levels persisted without significant humoral response. In order to better understand if central tolerance plays a role in this mechanism of neonatal tolerance, thymic subpopulations of OT2 transgenic mice that received no virus at birth were compared to OT2 mice crossed with CAG-OVA mice (allowing the earliest post-conception exposure to membrane-bound OVA). There was significant reduction of CD4+ positive cells (81.3% vs. 10.1%, p <0.001) suggesting that early exposure to OVA led to negative selection during immunologic ontogeny. However, OT2 mice that received neonatally administered AAV-OVA demonstrated 69.6% CD4+ thymocytes as adults, suggesting that thymic deletion has little if any role in the mechanism of neonatal tolerance.