Adeno-associated Virus Vectors May Induce Innate Immune Signaling Pathways

Adeno-associated virus (AAV)-based viral vectors used in human gene therapy can induce innate immune pathways, leading to the initiation of the body's adaptive immune response. Recently, in a review article entitled "Innate Immune Sensing of Adeno-Associated Virus Vectors" published in the international journal Human Gene Therapy, scientists from Indiana University and other institutions described the range of possible redundant innate immune pathways that AAV vectors can activate, which will lead to excessive adaptive immune responses.

Researcher Roland Herzog said that they described the body's innate immunity and understanding of viral vectors, including sensing the viral genome through toll-like receptor 9 and toll-like receptor 2 as capsid sensors. At the same time, the researchers also discussed the key role of interleukin-1-receptor-1 in B cell and T cell activation, cytoplasmic DNA sensors, and cytoplasmic RNA sensors in the article. The researchers analyzed cells that mediate innate sensing, immunity, and the transition to adaptive CD8+ T cell responses.

TLR9 was the first innate sensor identified as critical for AAV innate sensing. Using mouse models and in vitro cultures of human peripheral blood mononuclear cells (PBMCs), Zhu et al. demonstrated that pDCs, but not conventional DCs (cDCs) or macrophages, respond to the AAV genome via the TLR9-MyD88 pathway to produce IFN I, thereby promoting adaptive immune responses against the AAV capsid as well as the AAV-encoded transgene product. These results came as a surprise to the AAV community as AAVs are known to be poor transducers of antigen-presenting cells (APCs). However, it is possible that AAVs can be endocytosed by APCs, leading to capsid degradation by endosomal proteases, thereby exposing the AAV genome to endosomal TLR9. Since this initial discovery, the TLR9-MyD88 pathway has emerged as an essential component of AAV innate immune sensing, driving early innate inflammation and IFN I production in tissues, promoting CD8+ T cell activation, and altering antibody responses. A recent study found that CpG motifs in the vector genome promote the emergence of a pDC-like myeloid cell population in mice that directly binds antibody transgene products via Fc-FcγR interactions, thereby reducing the early expression of vector-encoded immunoglobulins.

In a murine model of AAV liver gene transfer, Martino et al. demonstrated increased TLR2 expression shortly after the administration of scAAV vectors. Using primary cultures of human nonparenchymal cells, Hosel et al. demonstrated that both liver sinusoidal endothelial cells and Kupffer cells (KCs) upregulated TLR2 and induced the cytokines IL-1β, IL-6, IL-8, and TNF-α upon sensing AAV capsids. A recent study found that TLR2 deficiency had a mild effect on CD8+ T cell and antibody production against transgene products in mouse liver gene transfer, resulting in slightly increased levels of circulating transgene products. However, adaptive immune responses to capsids were found to be TLR2-independent in a mouse muscle gene transfer model. It is important to emphasize that upregulation of various innate sensors and cytokines is mediated by cytokines such as IFN I, and therefore, pure upregulation may not represent direct involvement. To date, a strong dependence of immune responses to AAV gene transfer on TLR2 has not been documented.

Figure 1. Multiple innate sensing pathways mediate cellular responses to AAV-encoded transgene product.

Although AAV vectors contain weaker inflammatory signals than many other viruses or vector systems, they still elicit an innate response that has major implications for gene therapy. Insights into the mechanisms of the innate immune response to AAV have led to the design of vectors with lower immunogenicity and the development of superior immunosuppression regimens. Innate immunity is associated with adaptive responses and immunotoxicity. Therefore, further investigation of innate immunity to AAV remains critical.

More information is needed on target tissue effects and routes of administration. Additional pathways may be identified, and redundancy depending on tissue and vector dose may require bypassing or blocking more than one pathway in certain applications. It may not be possible to completely prevent an adaptive response by blocking innate signals. For example, the signaling requirements to reactivate a memory response are often lower than those for a primary response. Animal models help identify pathways but can never fully recapitulate human therapy, as shown by the various inconsistent immune responses to AAV vectors: CD8+ T cell responses to the capsid, complement activation and associated toxicity, the effects of pre-existing immunity from natural infection, or cross-reactivity of antibodies against capsid formation.

Reference

Cao D, et al. Innate Immune Sensing of AAV Vectors. Human Gene Therapy, 2024 (ja).