Adeno-associated viral (AAV) vector-based gene transfer is a promising strategy to express transgenes to supplant defective endogenous protein function and/or modulate the course of disease. Today, five AAV-based products have been FDA approved and intense pre-clinical and clinical investigations are underway for their application to a broad range of diseases involving diverse tissues and systems [1].

Long-term, effective transgene expression can be antagonized by the induction of inflammatory B and T lymphocytes that recognize AAV capsid and/or transgene [2]. A long-known source of inflammation for facilitating lymphocyte activation are unmethylated CpG dinucleotides, which are present in variable abundance in natural and recombinant AAV genomes [3]. Unmethylated CpGs serve as bacteria- or virus-derived pathogen-associated molecular pattern ligands for Toll-like receptor (TLR)-9 [4], [5]. TLR9 is a pattern recognition receptor (PRR) that usually resides in the endosomal compartment and is principally expressed in B cells and plasmacytoid dendritic cells (pDCs) [6]. CpG-TLR9 stimulation results in the MyD88 adaptor-mediated activation of NF-κB and IRF7 to effect an innate response marked by the expression of diverse inflammatory cytokines and chemokines, including the Type-I interferons, TNF-α, IL-1β, IL-6, and CCL2 [7]. These factors in turn promote activation of B cells and T cells against AAV capsid and transgene product.

AAV1-human Factor IX (hFIX)administration with the TLR9 agonist oligodeoxynucleotide 1826 (ODN1826) in mice resulted in reduced hFIX expression and significant induction of anti-FIX antibody when compared to sole AAV-hFIX administration [8]. This associated with early (24 h) activation of lymph node and splenic conventional DCs (cDCs), pDCs, and B cells, largely manifested as upregulation of the co-stimulatory molecules CD40 and CD86. Further study noted the importance of Treg cells towards shaping antibody formation to transgene product [9]. In an independent study, CpG motif depletion in a FIX-expressing construct led to reduced neutralizing antibody levels to AAV8 capsid [10].

The impact of CpG-TLR9 signaling on T cell activation following AAV-mediated gene transfer has also been explored. Mice deficient in TLR9 expression showed reductions in nuclear beta-galactosidase (nLacZ) transgene-specific T cell proliferation and IFN-gamma (IFNγ) expression subsequent to AAVrh32.33nLacZ administration [11]. These mice exhibited improved nLacZ expression that associated with reduced inflammatory gene expression and decreased MHC-II expression in transduced muscle. Depleting CpGs in the vector led to similar observations in multiple studies [12], [13], [14], [15], implying that during in vivo AAV-mediated gene transfer, pDCs are stimulated to produce Type-I IFN after TLR9 recognition of CpGs present on the vector genome.

AAV vectors can be used to deliver therapeutic antibodies, targeting a wide range of pathologies [16], [17], [18]. We have been interested in designing vectors able to produce antibodies directed against elements of the contact system such as kallikrein (anti-plasma kallikrein antibodies, α-pKal Ab) for the treatment of hereditary angioedema (HAE) [19], [20]. While evaluating the kinetics of human α-pKal Ab expression from an AAV9 vector in mice, we confirmed that CpG depletion of the human antibody transgene sequence benefited expression in the long-term. Unexpectedly, we also observed a significant effect of CpG depletion as early as 24–48 h following vector administration. We show that a PDCAlo/−CD11b+ myeloid cell population that can bind to the antibody encoded by the transgene is amplified in the presence of CpGs and contributes to the early reduction of transgene product levels.