TLR9-Activating CpG-B ODN but Not TLR7 Agonists Triggers Antibody Formation to Factor IX in Muscle Gene Transfer

Abstract

Innate immune signals that promote B cell responses in gene transfer are generally ill-defined. In this study, we evaluate the effect of activating endosomal Toll-like receptors 7, 8, and 9 (TLR7, TLR7/8, and TLR9) on antibody formation during muscle-directed gene therapy with adeno-associated virus (AAV) vectors. We examined whether activation of endosomal TLRs, by adenine analog CL264 (TLR7 agonist), imidazolquinolone compound R848 (TLR7/8 agonist), or class B CpG oligodeoxynucleotides ODN1826 (TLR9 agonist), could augment antibody formation upon intramuscular administration of AAV1 expressing human clotting factor IX (AAV1-hFIX) in mice. The TLR9 agonist robustly enhanced antibody formation by the 1st week, thus initially eliminating systemic hFIX expression. By contrast, the TLR7 and TLR7/8 agonists did not markedly promote antibody formation, or significantly reduce circulating hFIX. We concurrently investigated the effects of these TLR agonists during muscle gene transfer on mature B cells and dendritic cells (DCs) in the draining lymph nodes including conventional DCs (CD11b + or CD8a + cDCs), monocyte-derived dendritic cells (moDCs), and plasmacytoid dendritic cells (pDCs). Only TLR9 stimulation caused a striking increase in the frequency of moDCs within 24 h. The TLR7/8 and TLR9 agonists activated pDCs, both subsets of cDCs, and mature B cells, whereas the TLR7 agonist had only mild effects on these cells. Thus, these TLR ligands have distinct effects on DCs and mature B cells, yet only the TLR9 agonist enhanced the humoral immune response against AAV-expressed hFIX. These new findings indicate a unique ability of certain TLR9 agonists to stimulate B cell responses in muscle gene transfer through enrichment of moDCs.

Immune Responses to Viral Gene Therapy Vectors

“…Studies in mice demonstrated a unique ability of TLR9 agonists to activate antibody responses to the transgene product in muscle gene transfer, which occurred through induction of moDC responses that enhance activation of T follicular helper T cells. 73 , 74 Therefore, moDC activation is a driver of T cell responses that promote antibody formation. Complement may also be involved in NAB formation.…”

Immune Response Mechanisms against AAV Vectors in Animal Models

“…A recent study found that co-administration of a TLR7 agonist (to mimic dsRNA sensing) along with IM delivery of a ssAAV vector failed to reduce transgene product levels in mice, raising doubts about whether dsRNA sensing is relevant in priming AAV vector and transgene product adaptive immune responses. 17 Thus, the role of dsRNA in the overall innate sensing of AAV vectors remains unclear and requires further study.…”

A Molecular Revolution in the Treatment of Hemophilia

“…78 The RNA backbone of fitusiran contains chemical modifications (including 2′-deoxy-2′-fluoro, and 2′-O-methyl substitutions in ribonucleotides and undisclosed replacements of phosphodiester linkages between them) that prevent its degradation by nucleases and recognition by Toll-like receptor (TLR)3 and TLR7, which are innate immune receptors sensing double-stranded RNAs. [78][79][80][81] Also, the siRNA is conjugated to triantennary Nacetylgalactosamine (GalNAc), which mediates its uptake by hepatocytes through asialoglycoprotein receptor (ASGPR), abundantly expressed in the liver. 78,82 The antisense strands of siRNAs are stable within the RISC for weeks, so the same siRNA molecule may target multiple transcripts, which produces a durable knockdown, and so therapeutic effect (fitusiran is dosed once monthly in the ongoing trials).…”