Recombinant AAV (rAAV) vectors are a key component of an emergent therapeutic paradigm with a demonstrated definitive benefit for genetic diseases. Immune responses are among the most challenging barriers to human gene therapy. Viral vectors are highly advantageous because of their evolved ability to negotiate complex intracellular pathways to efficiently deliver a DNA payload; however, the human immune system has evolved multiple pathways, including innate pathogenassociated molecular pattern (PAMP) sensors that trigger adaptive effector functions to eliminate infected cells. 1 While rAAV product developers cannot avoid using AAV capsid, which is the source of viral peptides that render transduced cells targets for capsidspecific cytotoxic T lymphocytes (CTLs), the elimination of immune co-stimulatory features is important. Unmethylated CpG dinucleotide-based motifs (CpGs) are known PAMPs that bind and dimerize monomeric TLR9 expressed in human dendritic cells 2, 3 and have been shown to cause activation of the Toll-like receptor (TLR) 9-MyD88 signaling pathway, thereby promoting CTL responses to AAV vectors in non-clinical models. 4–6 Polynucleotides containing unmethylated CpGs are adjuvants used in vaccine development to stimulate strong cellular immune responses. 7 Details now available for eight hemophilia B gene therapy trials that used differing codon-modification strategies resulting in a broad range of CpG content (0-to 5-fold of wild type) in the factor IX (FIX) open reading frame (ORF) reveal that low CpG correlates strongly to long-term expression. Herein is provided a perspective that unmethylated CpG content in AAV vectors is the “key” attribute that triggers transgene expression-limiting immune responses in humans and that novel clinical vector production strategies to increase CpG methylation should be developed.

Clinical Evidence A discussion of the role of CpGs in AAV vectors and their contribution to immunotoxicity and loss of transgene expression in hemophilia gene therapy was catalyzed by the report by Chapin and colleagues (J. Chapin et al., 2018, 14th Workshop on Novel Technologies and Gene Therapies for Hemophilia, conference), which reported unexpected loss of FIX expression in 7 of 8 patients in their clinical trial using AAV8-FIXsc (Padua) investigational product BAX335. They hypothesized that CpG enrichment resulting from “codon-optimization” of the FIX ORF was the root cause of the CTL formation that eliminated transduced cells. This hypothesis is supported by results from seven other AAV-based gene therapy trials for hemophilia B reporting long-term follow-up, as summarized in Table 1. Among the variables, including serotype, expression cassette configuration, production method, vector genome (vg) and estimated total capsid dose, and the use of immune-suppression, low CpG content is the only parameter that fully correlates with long-term FIX expression. Codon modification was used to remove the 19 CpGs present in wild-type FIX cDNA in all four trials that reported durable FIX expression in all (33 combined) subjects, as well as the absence of or modest CTL responses that were easily controlled by transient immune suppression. In contrast, a different codon modification approach that aimed to increase the translational kinetics of the expression cassette and, in the process, increasing CpGs by approximately 5-fold over wild-type cDNA, was used in the three