Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses

Abstract

Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome. The engineered vectors elicited markedly reduced innate immune and T cell responses and enhanced gene expression in clinically relevant mouse and pig models across different tissues, including liver, muscle, and retina. Subretinal administration of higher-dose AAV in pigs resulted in photoreceptor pathology with microglia and T cell infiltration. These adverse findings were avoided in the contralateral eyes of the same animals that were injected with the engineered vectors. However, intravitreal injection of higher-dose AAV in macaques, a more immunogenic route of administration, showed that the engineered vector delayed but did not prevent clinical uveitis, suggesting that other immune factors in addition to TLR9 may contribute to intraocular inflammation in this model. Our results demonstrate that linking specific immunomodulatory noncoding sequences to much longer therapeutic nucleic acids can “cloak” the vector from inducing unwanted immune responses in multiple, but not all, models. This “coupled immunomodulation” strategy may widen the therapeutic window for AAV therapies as well as other DNA-based gene transfer methods.

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

“…In case of editing the promoter, one would have to empirically determine which sequences can be changed and what to change them to without altering promoter strength or specificity. An alternative approach to eliminate TLR9 signaling is to include TLR9 inhibitory DNA sequences in the vector construct, as recently been shown by Chan et al (54). However, it should be pointed out that even TLR9 deficient mice do not show complete abolishment of CD8 + T cell responses, as our current and published studies have shown (7)(8)(9).…”

Challenges Posed by Immune Responses to AAV Vectors: Addressing Root Causes

“…If achievable, methylation of CpG motifs to ‘humanize’ this epigenic attribute is predicted to reduce TLR9 activation. Another approach is the incorporation of TLR9 inhibitory nucleotides sequences into the AAV expression cassette ( 60 ).…”

Overcoming Immunological Challenges Limiting Capsid-Mediated Gene Therapy With Machine Learning

“…ML-powered capsid design and engineering will transform the landscape of GT delivery modalities, however non-capsid improvements are also relevant from an immunological perspective and can also increase therapeutic effectiveness. Reducing the activation of innate immunity by engineering the vector genome (66,67), co-administration with targeted immune-modulators to induce tolerance toward the vector (68) or depletion of pre-existing anti-capsid antibodies (69) should work in synergy with engineered capsids to pave a path for repeat vector administration, while further increasing the safety and tolerability of next generation GTs.…”