Effect of CpG Depletion of Vector Genome on CD8+ T Cell Responses in AAV Gene Therapy

Abstract

Adeno associated viral (AAV) vectors have emerged as a preferred platform for in vivo gene replacement therapy and represent one of the most promising strategies to treat monogenetic disorders such as hemophilia. However, immune responses to gene transfer have hampered human gene therapy in clinical trials. Over the past decade, it has become clear that innate immune recognition provides signals for the induction of antigen-specific responses against vector or transgene product. In particular, TLR9 recognition of the vector’s DNA genome in plasmacytoid dendritic cells (pDCs) has been identified as a key factor. Data from clinical trials and pre-clinical studies implement CpG motifs in the vector genome as drivers of immune responses, especially of CD8+ T cell activation. Here, we demonstrate that cross-priming of AAV capsid-specific CD8+ T cells depends on XCR1+ dendritic cells (which are likely the main cross-presenting cell that cooperates with pDCs to activate CD8+ T cells) and can be minimized by the elimination of CpG motifs in the vector genome. Further, a CpG-depleted vector expressing human coagulation factor IX showed markedly reduced (albeit not entirely eliminated) CD8+ T cell infiltration upon intramuscular gene transfer in hemophilia B mice when compared to conventional CpG+ vector (comprised of native sequences), resulting in better preservation of transduced muscle fibers. Therefore, this deimmunization strategy is helpful in reducing the potential for CD8+ T cell responses to capsid or transgene product. However, CpG depletion had minimal effects on antibody responses against capsid or transgene product, which appear to be largely independent of CpG motifs.

Efficacy of AAV serotypes to target Schwann cells after intrathecal and intravenous delivery

“…This is in contrast to CNS studies showing that AAVrh10 provides higher expression compared to other serotypes 34 , 35 . This might be either due to the fact that a number of capsids are trapped in different cell types such as ependymal cells, or due to the presence of unproductive viral particles which may result also in CD8 toxicity associated to TLR9 activation 38 , 39 . In contrast to expression rates, expression levels of EGFP and Cx32 detected by immunoblot analysis tended to be higher with AAVrh10 compared to AAV9 although there was marked variability between samples from different animals.…”

Immunogenicity and toxicity of AAV gene therapy

“…The most practiced approach has been removal of CpG rich sequences from the AAV vectors' genome. Animal studies have shown that a reduction of the vectors' CpG motifs blunts CD8 + T cell responses without modifying B cell responses (52). The ITRs are very rich in CpG sequences that cannot be modified so that complete CpG depletion of the AAV vectors' genome may not be feasible (54).…”

Gene Therapy Developments for Pompe Disease

“…These innate problems associated with rAAV need to be carefully studied in order to improve overall outcomes. The pivotal role of TLR9 activation [ 93 ] as a mediator of both the cellular and humoral immune response needs to be analyzed further, as well as CpG motifs in the vector genome, which can drive the immune responses associated with CD8+ T-cell activation [ 94 ]. The introduction of novel technologies, such as incorporating short DNA oligonucleotides in vector genomes that antagonize TLR9 activation, may be beneficial in reducing TLR9-mediated immune responses and may allow higher doses to be infused in patients [ 95 ].…”