Research ArticleGene Therapy

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

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Science Translational Medicine  10 Feb 2021:
Vol. 13, Issue 580, eabd3438
DOI: 10.1126/scitranslmed.abd3438

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Cloaking vector genomes

Adeno-associated viral (AAV) vectors are frequently used in gene therapy, but they trigger an immune response through Toll-like receptor 9 (TLR9), a receptor that senses foreign DNA. Chan and colleagues incorporated short noncoding DNA sequences into the AAV genome to directly antagonize TLR9 activation and “cloak” the much larger AAV DNA sequence from detection. In mice and pigs, administration of these modified AAV vectors resulted in reduced innate immune and T cell activation with improved gene expression. In macaques, intravitreal injection of the modified vector delayed but did not fully prevent development of uveitis. Incorporation of these cloaking sequences may improve the success of AAV-based gene therapies.


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.

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