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.

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.

Adeno-Associated virus (AAV) vectors have become an invaluable tool in research and the clinic. Likewise, AAV is used in various animal models for investigating a host of diseases. However, AAV shedding, or the release of AAV particles from the body, remains an understudied field which could be detrimental to researchers and be a potential contributor to pre-clinical variability. To our knowledge, no studies have been performed to evaluate any relationship between AAV shed particles and the effects these particles may have on cage mates. As such, we evaluated the level of AAV viral genomes in the fecal matter of C57BL6/J mice following the intravenous administration of AAV8. Here we show that AAV genomes are shed from female C57BL/6J mice for a minimum of 2 months following intravenously injected AAV8 vector. We also sought to determine if these shed AAV particles could affect non-vectored cage mates in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). We found an association between caging vectored animals with non-vectored animals and a reduction in disease severity in the non-vectored animals. We also found an association between disease severity and the ratio of vectored:non-vectored animals in the cage. Our findings should act as a word of caution to researchers performing AAV studies and should be considered to prevent confounding variables and bias in results.

Adeno-associated viral vectors (AAV) are the leading gene therapy in the clinic. AAV9 has been of particular interest due to its wide tropism for multiple tissue types as well as being able to cross the blood-brain barrier and transduce central nervous system tissues. However, effectively and safely targeting extrahepatic tissue following the systemic administration of AAV9 remains a challenge due to high rates of liver transduction and liver toxicity. Thus, a crucial first step in developing a safe AAV9-based vector is to reduce liver targeting. Here we utilized rational design techniques to make five point mutations in the AAV9 capsid. In doing so, we developed a novel AAV9 variant, AAV9-DM, that is characterized by reduced liver tropism as compared to AAV9 and other liver de-targeted AAV9 mutants. We show that AAV9-DM is effective at transducing cells in vivo, resulting in robust transgene expression over a 9-week period. Importantly, the AAV9-DM capsid maintains the ability to transduce non-hepatic tissues with a biodistribution similar to AAV9. This new mutant represents a novel AAV capsid that may be the basis for developing safer therapeutics to target extrahepatic tissue while reducing adverse side effects related to liver transduction.