The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver

In mice, 1e11 vg scAAV8.FIX given systemically caused moderate liver type I interferon gene expression at 2 hpi, the peak time.

“systemic administration of 1 × 1011 vector genomes (vg) (equivalent to ~4 × 1012 vg/kg, a therapeutically relevant dose in the clinic) of scAAV8.FIX in mice stimulated moderate type I interferon (Ifnb1 and Ifna13) gene expression in the liver at 2 hours post-injection (hpi), the peak time point as previously described (16).”

The AAV vector genome triggers TLR9, a receptor that detects foreign DNA.

“the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA”

The innate response to self-complementary AAV in mice required TLR9/MyD88.

“We confirmed with Tlr9−/− and Myd88−/− mice that the innate immune response to self-complementary AAV was indeed dependent on the TLR9/MyD88 pathway (Fig. 1D and 1E), as previously shown in the literature (15–17).”

The TLR9-initiated pathway is a well-recognised component of the pro-inflammatory response to AAV-mediated gene therapy in the retina and elsewhere in the body.

“The TLR9-initiated pathway is a well-recognised component of the pro-inflammatory response to AAV-mediated gene therapy in the retina (Chan et al., 2021b) and elsewhere in the body (Zhu et al., 2009; Martino et al., 2011; Faust et al., 2013).”

scAAV heightens innate immune response via enhanced activation of TLR9/MyD88 signaling in a dose-dependent manner with induction of IL-6, TNF-α, and other proinflammatory cytokines.

“Notably, scAAV heightens innate immune response via enhanced activation of the TLR9/MyD88 signaling in a dose-dependent manner with induction of IL-6, TNF-α, and other proinflammatory cytokines [13, 14], suggesting increased immunogenicity associated with scAAV.”

Self-complementary AAV vectors exhibited an increased innate immune response to the transgene compared with single-stranded AAVs, possibly due to enhanced activation of the TLR9/MyD88 signaling cascade.

“In addition, other studies have shown that self-complementary AAV (scAAV) vectors exhibited an increased innate immune response to the transgene compared with single-stranded AAVs (ssAAVs), possibly due to enhanced activation of the TLR9/MyD88 signaling cascade [30, 44, 45].”

In some patients, these mechanisms can progress to a range of symptoms of varying severity, such as vomiting, acute liver failure, and cardiac and kidney injury (atypical hemolytic uremic syndrome) after AAV administration.

“In some patients, these mechanisms can progress to a range of symptoms of varying severity, such as vomiting, acute liver failure, and cardiac and kidney injury (atypical hemolytic uremic syndrome [aHUS]) after AAV administration (NCT03362502 and NCT03368742) [44].”

TLR9 inhibitory oligonucleotides restored the increased macrophage infiltration triggered by scAAV administration.

“For example, TLR9 inhibitory oligonucleotides restored the increased macrophage infiltration, triggered by scAAV administration [44].”

The difference in responses would likely be more pronounced if more immunogenic DNA were used, for instance CpG-rich or self-complementary.

“It is likely that the difference would be more pronounced if more immunogenic DNA would be used, for instance CpG-rich or self-complementary (65–69).”

TLR9 sensing of AAV vector genomes is required for anti-capsid CD8+ T cell responses.

“Importantly, TLR9 sensing of AAV vector genomes has been shown to be required for anti-capsid CD8+ T cell responses (9–14).”

CpG depletion does not eliminate antibody formation against the capsid or the transgene product, which can be modified by but does not depend on the TLR9-MyD88 pathway.

“Another limitation is that CpG depletion does not eliminate antibody formation against the capsid or transgene product, which can be modified by but does not depend on the TLR9-MyD88 pathway (9, 12, 15).”

The immunological cascades following TLR9 activation result in release of cytokines, inflammation, and cytotoxic T cell (CTL) responses.

“The subsequent immunological cascades ultimately results in release of cytokines, in fl ammation and cytotoxic T cell (CTL) responses (24, 25).”

The TLR9-MyD88 pathway is an important component of innate immune sensing of AAV, driving early innate inflammation and IFN I production in tissue, promoting CD8+ T cell activation, and modifying antibody responses.

“Since this initial discovery, the TLR9-MyD88 pathway has been consolidated as an important component of innate immune sensing of AAV, driving early innate inflammation and IFN I production in the tissue, promoting CD8 + T cell activation, and modifying antibody responses.”

These IL-1R1-dependent responses did not require inflammasomes, consistent with earlier observations that AAV vectors are ineffective in activating inflammasomes.

“Interestingly, these responses did not require inflammasomes, consistent with earlier observations that AAV vectors are ineffective in activating inflammasomes.”

In a murine model of AAV liver gene transfer, Martino et al. showed increased TLR2 expression soon after administration of scAAV vector.

“In a murine model of AAV liver gene transfer, Martino et al. showed increased TLR2 expression soon after administration with scAAV vector.”

Self-complementary AAV vectors may be more potent activators of TLR9-MyD88-mediated immune responses than single-stranded DNA genome vectors.

“However, these vectors may also be more potent in the activation of TLR9-MyD88 mediated immune responses than AAV vectors containing the ss-DNA genome.”

The configuration of the vector genome, in addition to CpG content, determines the magnitude of TLR9 signaling.

“Besides CpG contents, the configuration of the vector genome has been identified as a factor that determines the magnitude of TLR9 signaling.”

Various studies have reported upregulation and secretion of IL-1α and IL-1β cytokines in different disease models in the context of AAV gene therapy.

“In the context of AAV gene therapy, various studies have reported the upregulation and secretion of both IL-1a and IL-1b cytokines in different disease models.”

TLR9-mediated innate immune responses appear largely independent of AAV capsid.

“TLR9-mediated innate immune responses appear largely independent of AAV capsid.”

Multiple studies have identified TLR9-MyD88 signaling in response to the AAV genome as an innate PRR signal critical for activation of CD8+ T cells against capsid or genome-encoded antigens.

“multiple studies have identified TLR9-MyD88 signaling in response to the AAV genome as an innate PRR signal that is critical for activation of CD8+ T cells against capsid or genome-encoded antigens.22,23,24”

The previously documented rapid TLR9-dependent innate response in the liver that occurs within hours after AAV vector administration is not required for CD8+ T cell activation against the transgene product.

“Our new findings imply that the previously documented rapid TLR9-dependent innate response in the liver that occurs within hours after AAV vector administration is not required for CD8+ T cell activation against the transgene product.22”

Martino et al. originally noted that TLR9-dependent innate immune signaling was induced by the self-complementary rAAV genome.

“This has been the case since Martino et al. originally noted that TLR9-dependent innate immune signaling was induced by the self-complementary rAAV genome [91].”

scAAV vectors increase the innate response to the transgene compared to single-stranded AAV (ssAAV) vectors.

“However, these vectors also increase the innate response to the transgene compared to single-stranded AAV (ssAAV) vectors and may thus not be the most suitable approach [91,129].”

All of the AAV-induced cytokines and chemokines can be upregulated by activation of TLR9 in the periphery using synthetic CpG-rich oligonucleotides.

“all of the AAV-induced cytokines and chemokines can be upregulated by activation of TLR9 in the periphery using synthetic CpG-rich oligonucleotides.8,9,10,11,12,52,53,54,55,56,57,58”

TLR9 activation is an early and transient event in the immune response to the AAV genome.

“TLR9 activation is an early and transient event in the immune response to the AAV genome.9”