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Upon viral infection of the liver, CD8+T cell responses may be triggered despite the immune suppressive properties that manifest in this organ. We sought to identify pathways that activate responses to a neoantigen expressed in hepatocytes, using adeno-associated viral (AAV) gene transfer. It was previously established that cooperation between plasmacytoid dendritic cells (pDCs), which sense AAV genomes by Toll-like receptor 9 (TLR9), and conventional DCs promotes cross-priming of capsid-specific CD8+T cells. Surprisingly, we find local initiation of a CD8+T cell response against antigen expressed in ∼20% of murine hepatocytes, independent of TLR9 or type I interferons and instead relying on IL-1 receptor 1-MyD88 signaling. Both IL-1α and IL-1β contribute to this response, which can be blunted by IL-1 blockade. Upon AAV administration, IL-1-producing pDCs infiltrate the liver and co-cluster with XCR1+DCs, CD8+T cells, and Kupffer cells. Analogous events were observed following coagulation factor VIII gene transfer in hemophilia A mice. Therefore, pDCs have alternative means of promoting anti-viral T cell responses and participate in intrahepatic immune cell networks similar to those that form in lymphoid organs. Combined TLR9 and IL-1 blockade may broadly prevent CD8+T responses against AAV capsid and transgene product.

Targeted delivery and cell-type-specific expression of gene editing proteins in various cell types in vivorepresent major challenges for all viral and non-viral delivery platforms developed to date. Here, we describe the development and analysis of AVIDs, an engineered adenovirus-based gene delivery platform, that allows for highly targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells (HSPCs) in vivoafter intravenous vector administration. Due to a set of refined structural modifications, intravenous administration of AVIDs did not trigger cytokine storm, hepatotoxicity, or thrombocytopenia. Single intravenous administration of AVIDs to humanized mice, grafted with human CD34+cells, led to up to 20% transduction of CD34+CD38-CD45RA-HSPC subsets in the bone marrow. Importantly, targeted in vivotransduction of CD34+CD38-CD45RA-CD90-CD49f+subsets, highly enriched for human HSCs, reached up to 19%, which represented a 1900-fold selectivity in gene delivery to HSC-enriched over lineage committed CD34-negative cell populations. Because the AVID platform allows for regulated, cell type-specific expression of gene editing technologies as well as expression of immunomodulatory proteins to ensure persistence of corrected HSCs in vivo, the HSC-targeted AVID platform may enable development of curative therapies through in vivogene correction in human HSCs after a single intravenous administration.