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Review
. 2021 Jan 4;131(1):e143780.
doi: 10.1172/JCI143780.

Overcoming innate immune barriers that impede AAV gene therapy vectors

Manish Muhuri  1   2   3 , Yukiko Maeda  1   3   4 , Hong Ma  1 , Sanjay Ram  5 , Katherine A Fitzgerald  6 , Phillip Wl Tai  1   2   3 , Guangping Gao  1   2   7
Affiliations
Review

Overcoming innate immune barriers that impede AAV gene therapy vectors

Manish Muhuri et al. J Clin Invest. .

Abstract

The field of gene therapy has made considerable progress over the past several years. Adeno-associated virus (AAV) vectors have emerged as promising and attractive tools for in vivo gene therapy. Despite the recent clinical successes achieved with recombinant AAVs (rAAVs) for therapeutics, host immune responses against the vector and transgene product have been observed in numerous preclinical and clinical studies. These outcomes have hampered the advancement of AAV gene therapies, preventing them from becoming fully viable and safe medicines. The human immune system is multidimensional and complex. Both the innate and adaptive arms of the immune system seem to play a concerted role in the response against rAAVs. While most efforts have been focused on the role of adaptive immunity and developing ways to overcome it, the innate immune system has also been found to have a critical function. Innate immunity not only mediates the initial response to the vector, but also primes the adaptive immune system to launch a more deleterious attack against the foreign vector. This Review highlights what is known about innate immune responses against rAAVs and discusses potential strategies to circumvent these pathways.

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Conflict of interest statement

Conflict of interest: GG is a scientific cofounder of Voyager Therapeutics and Aspa Therapeutics, and holds equity in these companies. GG is an inventor on patents with potential royalties licensed to Voyager Therapeutics, Aspa Therapeutics, and other biopharmaceutical companies. PWLT is an inventor on patents with potential royalties licensed to Kanghong Therapeutics (patent application no. PCT/US2020/049243). KAF is an SAB member at Generation Bio and holds equity in this company.

Figures

Figure 1
Figure 1. Detection of AAV vector elements by PRRs.
rAAV capsids can activate TLR2 on the cell surface or endosomal membrane, which subsequently recruits MyD88 and phosphorylates IRAKs. rAAV capsids may break open in the endosome or lysosome and expose the genome to TLR9. Upon binding DNA, TLR9 activates the MyD88/IRAK pathway to induce proinflammatory cytokines like TNF-α and IL-6 (38). Vector genomes may also become exposed within the cytosol. Alternatively, rAAV-mediated stress may release mitochondrial DNA (mtDNA) to activate cytosolic DNA sensors. Upon binding DNA, cGAS promotes cGAMP synthesis, which activates the STING/IRF3 pathway and upregulates type I IFNs (146). IFNs and cytokines induce expression of ISGs and antiviral responses. IFI16 can promote cGAS-mediated production of cGAMP to activate STING. Upon binding DNA, AIM2 and IFI16 form the inflammasome and promote maturation of IL-1β and IL-18 (82). Nuclear-localized rAAV genomes may also activate cGAS and IFI16. Nuclear cGAS can induce IFN-β production by stabilizing IFI16 (84), which can be exported into the cytoplasm to activate the inflammasome pathway (147). IFI16 also silences viral gene expression in the nucleus (76). AIM2 is also found in the nucleus (85), but it is not known whether AIM2 can sense viral DNA in nuclei. The cytosolic RNA sensors RIG-I and MDA5 can recognize RNA transcripts from rAAV and activate the TBK/IRF pathway to induce IFNs (88, 89, 92). Pathways that are only speculated to be involved are indicated by question marks. Pathways only implicated by circumstantial evidence are indicated with dashed arrows.
Figure 2
Figure 2. Complement activation by AAVs.
Antibodies bound to AAV particles are recognized by the complement protein C1 complex. When high doses of AAV are administered, AAV-antibody complex activates the classical pathway of complement, eventually leading to the formation of the membrane attack complex (MAC) (105). The target of the MAC ring during AAV infection is unclear. When low AAV doses are administered, C3b can bind to the AAV capsid, where it is converted to iC3b and subsequently to C3d by factor I and other cofactors. Cleavage fragments of C3 opsonize the target structure and serve as bridging molecules with receptors on the surface of the phagocytes. CR1 and CR3 expressed on the macrophage surface interact with C3b- or iC3b-opsonized AAV particles, leading to phagocytosis and macrophage activation. CR3 interaction with iC3b-opsonized AAV virions on DC surfaces also results in endocytosis and antigen presentation to naive T cells. C3d-bound AAVs can be recognized by CR2 on B cell surfaces. Co-ligation of CR2 with B cell receptor (BCR) results in augmented signaling that effectively lowers the threshold for B cell clonal expansion. Alternatively, DCs can also trap the C3d-opsonized AAV via CR2 and present the antigen to naive or previously antigen-engaged B cells during the processes of affinity maturation, isotype switching, and the generation of effector and memory B cells.
Figure 3
Figure 3. The role of APC-mediated immune responses toward AAV vectors.
(A) The mechanism of DC activation by rAAV. Vector genome sensing by TLR9 in pDCs’ endosomes triggers the activation of the TLR9/MyD88 signaling pathway that culminates in pDCs producing type I IFNs that directly signal to immature cDCs. This signaling event is required for effective priming and leads to activation and licensing of immature cDCs to mature cDCs. Licensing of cDCs enhances their ability to activate T cells. Activated cDCs also interact with CD4+ T cells, which may additionally contribute to licensing the cDCs to activate rAAV capsid–specific CD8+ T cells. Activated CD4+ Th cells also promote antibody formation against the rAAV capsid. (B) Depiction of rAAV-specific antigen presentation by APCs. Upon entry of AAV virions into cells by endocytosis, rAAV capsids can either be degraded in lysosomes or escape into the cytoplasm. Transcription and translation of the vector genome in the nucleus generate transgene proteins that, along with viral capsids, can be ubiquitylated and degraded in the proteasome into small peptides. These peptides are transported into the Golgi/endoplasmic reticulum by transporter associated with antigen presentation (TAP), loaded onto the MHC class I molecule, and presented on the surface of the target cell. This causes the cell to be recognized by a CD8+ T cell and, finally, eliminated by a capsid-specific CTL response. Capsid peptides are also loaded onto MHC class II molecules for presentation to CD4+ T cells for subsequent B cell activation and antibody production.
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