Liver-directed AAV gene therapy represents a unique treatment modality for a host of diseases. This is due, in part, to the induction of tolerance to transgene products. Despite the plethora of recognized regulatory cells in the body, there is currently a lack of literature supporting the induction of non-CD4+ regulatory cells following hepatic AAV gene transfer. In this work, we show that CD8+ regulatory T cells are up-regulated in PBMCs of mice following capsid only and therapeutic transgene AAV administration. Further, we demonstrate that hepatic AAV gene transfer results in a significant increase in CD8+ regulatory T cells following experimental autoimmune encephalomyelitis induction. Notably, this response occurred only in therapeutic vector treated animals, not capsid only controls. Understanding the role these cells play in treatment efficacy will result in the development of improved AAV vectors that take advantage of the full gamut of regulatory cells within the body.

Multiple Sclerosis is a demyelinating autoimmune disease brought on by self-reactive, myelin-specific T effector cells. Immunosuppressants are the most utilized tool to combat disease progression; however, they bring unwanted side effects. We have previously published on a novel gene immunotherapy that uses an adeno-associated virus (AAV) containing the full sequence of the myelin oligodendrocyte glycoprotein (MOG) that restores tolerance through liver-directed gene therapy, resulting in the generation of antigen-specific T regulatory cells (Tregs) that can ameliorate Experimental Autoimmune Encephalomyelitis (EAE). While the data is promising, the mechanism of this therapy remains enigmatic. To begin defining the mechanism initially, we cultured MOG-specific CD4 +T cells from 2D2 mice with liver cells from B6 mice that received either AAV.MOG or a saline control. We found a significant increase in the frequency of CD4 +CD25 +FoxP3 +Tregs when 2D2 T cells were cultured with liver cells from AAV.MOG-vectored mice in a 1:2 ratio compared to the control. Next, we injected 2D2 mice with AAV.MOG and isolated CD4 +CD25 +T cells 2 weeks later. To evaluate their suppressive ability they, or control cells, were adoptively transferred to recipient naïve B6 mice. After 24 hours, active EAE was induced. By day 15, disease severity in mice that received AAV.MOG donor 2D2 T cells was significantly reduced compared to the controls (CD4 +CD25 +T cells from wildtype B6 mice given either AAV.MOG or AAV.AQP4) Overall, these results demonstrated that livers transduced with AAV.MOG can induce 2D2 Tregs in vitro and that CD4 +CD25 +T cells from vectored 2D2 mice reduce EAE severity. Supported by a grant from the NIH (R01AI128074) and the Children's Miracle Network

Recombinant adeno-associated viruses (rAAVs) have become one of the leading gene therapies for treating a variety of diseases. One factor contributing to rAAVs’ success is the fact that a wide variety of tissue types can be transduced by different serotypes. However, one commonality amongst most serotypes is the high propensity for liver transduction when rAAVs are administered peripherally. One of the few exceptions is the naturally occurring clade F AAV hematopoietic stem cell 16 (AAVHSC16). AAVHSC16 represents an interesting capsid in that it shows minimal liver transduction when injected peripherally. For capsids other than AAVHSC16, targeting non-liver tissues via peripheral AAV injection represents a challenge due to the high liver transduction. Thus, there is a demand for liver-de-targeted rAAV vectors. The rational design of rAAV capsids relies on current knowledge to design improved capsids and represents one means of developing capsids with reduced liver transduction. Here, we utilized data from the AAVHSC16 capsid to rationally design four non-clade F rAAV capsids that result in reduced liver transduction following peripheral injection.