Adeno-associated virus (AAV) is a very promising gene therapy vector in pre-clinical and clinical trials. However, recent studies have demonstrated that the AAV2 capsid can induce a cytotoxic T lymphocyte (CTL) response via both classical antigen presentation and cross-presentation pathways, thereby raising concerns associated with immune response to AAV vectors. In particular, it has been suggested that capsid specific CTLs eliminated AAV2 transduced liver cells and resulted in therapeutic failure in a hemophilia B clinical trial. The goal of this proposal is to understand the mechanisms of presentation of AAV capsid antigens in vitro and in vivo and more importantly, to devise strategies to evade the immune response. Our long term goals are to enhance the safety and efficacy of AAV vectors through formulation of novel immune evasion strategies. Since data from animal models have contradicted clinical observations outlined above (possibly due to poor immunogenicity of the AAV capsid in mice), we have integrated a strong immune domain OVA epitope SIINFEKL into the VP3 protein of the AAV2 capsid (AAV2-OVA). Decreased transgene expression was seen in mice with memory OVA CTLs following liver transduction with AAV2-OVA vector. In the current proposal, we will use AAV2-OVA vector to investigate the kinetics and mechanisms of AAV capsid cross-presentation in transduced cells in vitro and in vivo (Aim 1 and 2). Through these studies, we expect to thoroughly characterize the CTL response to AAV capsid proteins in mice that more accurately represents data obtained in humans. After AAV vector binds on cell surface, via endosomal uptake, AAV2 capsids must uncoat enroute to the nucleus prior to vector genome transcription. This trafficking route suggests that antigen presentation of capsids after transduction may follow a classical MHC-class I pathways. Many viruses (For example herpes, EB) evade host immune response by synthesizing small peptides called viral proteins interfering with antigen presentation (VIPR), we propose that integration of VIPR into AAV capsid evade host CTL mediated elimination of AAV transduced target cells (Aim 3). By engineering VIPRs into AAV2 capsid proteins, we will ensure that antigen presentation will be attenuated only in AAV2 transduced cells without systemic side effects on the immune system (as would be the case with immunosuppressive drugs or application of regulator T cells). These experiments rely on predetermined domains in AAV capsid proteins for incorporation of VIPR domains. A timely understanding is critical for the continued use of AAV therapy under the current protocols (i.e. without immunosuppression addendums).

Public Health Relevance

Lay summary Adeno-associated virus (AAV) has been used in over 50 clinical trials and proves to be very promising for gene therapy, due to long-term therapeutic gene expression after delivery of AAV vectors. Recently, data from one clinical trial for hemophilia B suggested that AAV2 could induce an immune response and thus eliminate AAV2 infected liver cells. However, the results from a mouse model study did not support these findings. One explanation of these contradictory results could be the weak immunogenic activity of AAV capsids in mice. To more accurately mimic the clinical trial and establish an appropriate mouse model, we propose to insert a strong immunogen into AAV2 capsid and use these mutant AAV capsids to make recombinant virus. After injection of these viruses into mouse, we will investigate whether immunogen specific CTLs eliminate AAV2 transduced target cells in mice. Additionally, to reduce any immune response elicited by the AAV capsid, we will insert the Epstein-Barr virus product EBNA-1 into a non-essential region of the AAV capsid. We will test whether EBNA-1 will mediate inhibition of an immune response and prevent eradication of AAV infected cells. The long-term goals of this proposal are to critically evaluate the immune response to AAV-infected cells and to develop a novel AAV vector that will evade an immune response without compromising function, thus improving AAV as a gene therapy tool and enhancing its therapeutic value.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1-TAG-M (01))
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Park, Eun-Chung
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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