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.
Bibliographical noteFunding Information:
Support for this work comes from the Wyss Institute for Biologically Inspired Engineering of Harvard University (to Y.K.C. and G.M.C.) and NIH (RM1 HG008525 to G.M.C.; EY026158 to M.A.M.). S.K.W. was supported by a Howard Hughes Medical Fellowship. C.L.C. is an investigator of Howard Hughes Medical Institute. This work was also supported by the European Research Council Consolidator Grant (grant agreement no. 617432 to F.M.), Kentucky Lions Eye Research Endowed Chair (to M.A.M.), and grants from the National Eye Research Centre, UK (BRI 021 to A.D.D.) and The Underwood Trust (8064 to A.D.D.). The intravitreal NHP study was funded and executed by Ally Therapeutics, a venture capital- funded startup.
Copyright © 2021 The Authors.