THE SCIENCE

Adeno-associated viral vectors (AAV) are leading candidates for gene therapy. However, in Phase I clinical settings, immune responses toward the delivery vehicle or trangene product have compromised safety and long-term gene replacement success. Toll-like receptor 9 (TLR9) has the potential to recognize unmethylated CpG motifs in the therapeutic expression cassettes packaged in an AAV capsid and to induce a pro-inflammatory immune response. The inventors of NxGEN technology identified TLR9 as a critical element in immunoreactivity toward AAV associated antigens following intramuscular gene transfer, and the absence of TLR9 signaling resulted in suppressed Th1 (IFN-gamma) responses toward capsid and transgene antigen, minimal cellular infiltrate, and stable, enhanced transgene expression in target muscles. These findings were subsequently translated into a CpG-depleted AAV vector utilized for skeletal muscle gene transfer into WT mice. Both vectors contained a cytoplasmic lacZ open reading frame expressed from a mammalian-derived promoter flanked by AAV2 inverted terminal repeats (ITRs) and differed only in the abundance of CpG motifs: the AAVCpG+ vector contained a total of 324 CpGs, while the AAVCpG- vector total CpG content was 16. Muscle gene transfer experiments revealed that the AAVCpG- vector established long-term, enhanced transgene expression, evaded inflammatory T cell responses, and minimized infiltration of effector T cells. These findings show the remarkable ability of AAV gene therapy vectors built using NxGEN technology to escape the immune responses that have challenged the field of gene therapy for decades, leading to safe and effective gene transfer.

FIGURE 1: AAVCpG+ transduced muscle exhibits a progressive loss of detectable β-gal expression, while the muscle sections from CpG-depleted AAV-transduced mice display robust and stable transgene expression. To test the hypothesis that CpG-depleted AAV vectors would exhibit prolonged transgene expression, WT mice were injected intramuscularly with 1 x 10¹¹ GC of AAVrh32.33CpG+ (AAVCpG+) or AAVrh32.33CpG– (AAVCpG–) vectors and muscle was recovered on day 35 (A and B) and day 60 (C and D) and stained for X gal.