81. Downregulation of Immune Responsive Chemokines Are Associated AAV8-Mediated Transgene Immune Tolerance

Abstract

Recombinant adeno-associated virus (rAAV)-mediated liver transduction can induce transgene product-specific immune tolerance, which can be further extended to the systemic tolerance. Because of such tolerogenic properties and its physiological functions, the liver has been a preferred target organ for AAV- gene therapy. The hepatic regulatory T cells (Tregs) are regarded as the major factor that contributes the immune tolerance in the liver. Here, we first tested if AAV capsid plays a role in inducing systemic immune tolerance. To this end, we used highly immunogenic Ovalbumin (OVA) as a model antigen expressed under the direction of a strong ubiquitous chicken β-actin (CB) promoter and packaged the vector with both AAV1 and AAV8 capsids for in vivo delivery to C57BL/6 mice. Compared to AAV1, AAV8 achieved significantly higher OVA expression without IgG production, regardless of intravenous (IV) or intramuscular (IM) injection. In addition, IV and IM delivered AAV1. OVA both generated OVA-specific CD8+ T cells in spleen, while AAV8-mediated deliveries blunted CD8+ T cell response and, instead, provoked B- and T-Reg responses. In an attempt to explore other possible cellular mechanism(s) led to hepatic tolerogenic properties, we profiled expression of innate and adaptive immune responsive genes that are possibly associated with liver immune tolerance in the context of rAAV transduction. We revealed that rAAV1.CB. OVA injections significantly elevated IL1a and CXCL1 gene expression as compared to the PBS and rAAV8 groups. As rAAV8 is more liver tropic than rAAVl, we hypothesized that hepatic OVA expression is essential to inducing immune tolerance. We tested this hypothesis by creating rAAV8.CB. OVA vectors (+/-) the binding sites for the liver enriched miR-122 and IV dosing C57BL/6 mice with the vectors at 4 doses (1×109, 1010, 1011 and 1012 gcs/mouse). We confirmed that miR-122 indeed effectively inactivated OVA expression from rAAV8.CB. OVA(+)MiR-122BS transduced liver; however, rAAV8.CB. OVA(-) miR122BS but not rAAV1CBOVA achieved sustained OVA expression in a dose dependent manner without eliciting OVA specific IgG. Finally, considering that the route of rAAV administration may affect transgene expression and immunity, we IM injected C57BL/6 mice with AAV8.CB. OVA (+/-) miR122BS vectors and found that AAV8.CB. OVA (-)miR-122BS expressed OVA efficiently without OVA-specific IgG production. In contrast, just as rAAV1.CB. OVA did, rAAV8.CB. OVA(+) miR-122BS produced significant OVA-specific IgG without OVA expression. Interestingly, IM delivered rAAV8.CB. OVA(+) miR-122 group expressed significantly higher levels of CXCL1, Myeloperoxidase(Mpo) and CCL5 in liver, as compared the PBS and rAAV8OVA(-)miR-122 groups as detected by qRT-PCR. Our data suggest that the downregulation of CXCL1, Mpo, CCL5 and IL1α in the liver are involved in the immune tolerance to rAAV8 delivered transgene. Those cytokines may represent therapeutic targets against autoimmune diseases.