Innate immune sensing of adeno-associated virus vectors

May combine two claims.

Dampening IL-1R1 signaling had a mild effect on transgene-specific antibodies but not on capsid-specific antibodies.

“Interestingly, dampening IL-1R1 signaling had a mild effect on transgene-specific antibodies but not on capsid-specific antibodies.”

• cites [38] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

AAV capsids undergo proteosome-mediated degradation in the cytoplasm, which may expose packaged genomes to cytoplasmic DNA sensors.

“AAV capsids undergo proteosome-mediated degradation in the cytoplasm, which may expose the packaged genomes to cytoplasmic DNA sensors.”

• cites [43] sourceNot checked yet

  Not checked in the free run.

• cites [44] sourceNot checked yet

  Not checked in the free run.

• cites [45] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

At high doses, host antiviral factors such as innate sensors would detect AAV-associated molecular signatures, initiating immunological reactions that may cause immunotoxicity and render gene transfer ineffective.

“At such high doses, host antiviral factors such as innate sensors would readily detect molecular signatures associated with AAV or the infection process, thereby initiating a cascade of immunological reactions that may cause immunotoxicity and ultimately render gene transfer ineffective.”

May combine two claims.

TLR2 deficiency had a modest effect on CD8+ T cell and antibody formation against the transgene product in hepatic gene transfer in mice, resulting in somewhat elevated circulating transgene product.

“A recent study found that TLR2 deficiency had a modest effect on CD8 + T cell and antibody formation against the transgene product in hepatic gene transfer in mice, resulting in somewhat elevated levels of circulating transgene product.”

• cites [38] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The endosomal DNA sensor TLR9 recognizes the AAV genome and promotes early inflammatory responses and interferon expression.

“Sensing of the AAV genome by the endosomal DNA receptor toll-like receptor 9 (TLR9) promotes early inflammatory response and interferon expression.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Binding of AAV capsid to complement components likely further elevates B cell activation.

“Binding of AAV capsid to complement components likely further elevates B cell activation.”

May combine two claims.

Upregulation of innate sensors and cytokines can be mediated by cytokines such as IFN I, so mere upregulation may not indicate direct involvement.

“It should be emphasized that upregulation of various innate sensors and cytokines is mediated by cytokines such as IFN I, and therefore, a mere upregulation might not be indicative of direct involvement.”

May combine two claims.

At least two innate signaling pathways promote CD8+ T cell activation in AAV gene transfer: TLR9>MyD88>IFN I>IFNαR and IL-1α/β>IL-1R1>MyD88.

“In summary, at least two innate signaling pathways exist that promote the activation of CD8 + T cell responses in AAV gene transfer, namely TLR9>MyD88>IFN I>IFNaR and IL-1a/b>IL-1R1>MyD88.”

May combine two claims.

Evidence has been presented for activation of TLR2 by AAV capsid and for additional innate receptors for nucleic acids.

“Finally, evidence for activation of TLR2 by the capsid and of additional innate receptors for nucleic acids has been presented.”

May combine two claims.

At high systemic vector doses in humans and non-human primates, AAV vectors can trigger complement activation through classical and alternative pathways, leading to severe toxicities.

“At high systemic vector doses in humans and in non-human primates, AAV vectors can trigger complement activation, with contributions by classical and alternative pathways, leading to severe toxicities.”

May combine two claims.

Activated platelets subsequently contribute to the innate response and endothelial injury.

“The activated platelets subsequently contribute to the innate response and endothelial injury.”

May combine two claims.

Kuranda et al. showed induction of pro-inflammatory cytokines IL-6 and IL-1β upon exposure of human PBMCs to empty AAV capsid, but did not directly implicate TLR2.

“Although Kuranda et al. showed induction of pro-inflammatory cytokines IL-6 and IL-1b upon exposure of human PBMCs with empty AAV capsid, they did not directly implicate TLR2 for this pro-inflammatory milieu.”

• cites [37] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

AAV can induce cytokine expression in monocyte-derived dendritic cells, which in turn increases antibody formation.

“AAV can induce cytokine expression in monocyte-derived DCs, which in turn increases antibody formation.”

May combine two claims.

Although AAV is substantially less inflammatory than many other viral vectors, treatments may require high vector doses up to 10^14 vg/kg, particularly for systemic administration.

“Although AAV is substantially less inflammatory compared with many other viral vectors, treatments may require administration of high vector doses of up to 10 14 vg/ kg, in particular for applications that depend on systemic vector administration.”

• cites [9] sourceNot checked yet

  Not checked in the free run.

• cites [10] sourceNot checked yet

  Not checked in the free run.

• cites [11] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

At high vector doses, immune responses still occurred despite TLR9 inhibition; in macaques injected intravitreally, TLR9 inhibition merely delayed immune-mediated clinical uveitis.

“However, immune responses were still observed at high vector doses, for instance, in macaques injected through intravitreal route.TLR9 inhibition merely delayed immune-mediated clinical uveitis in these experimental animals.”

• cites [33] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Both IL-1α and IL-1β contribute to this IL-1R1-dependent CD8+ T cell response, with IL-1α perhaps having a greater contribution, and the response can be blunted by blocking IL-1 signaling.

“Both IL-1a and IL-1b contribute to this response (with IL-1a having a perhaps greater contribution), which can be blunted by blocking IL-1 signaling.”

• cites [38] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

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.”

• cites [23] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

TLR9-dependent reduction of dendritic length and complexity and disruption of synaptic transmission occurred in mouse somatosensory cortex after AAV gene transfer, accompanied by changes in AMPA receptor subunit composition and prevented by TLR9 blockade.

“Another recent investigation reports TLR9-dependent reduction of dendritic length and complexity and disruption of synaptic transmission in mouse somatosensory cortex in response to AAV gene transfer, which was accompanied by changes in AMPA receptor subunit composition but could be prevented by TLR9 blockade.”

• cites [28] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

In murine models and human PBMC cultures, plasmacytoid dendritic cells but not conventional dendritic cells or macrophages respond to the AAV genome through the TLR9-MyD88 pathway, producing IFN I that promotes adaptive immune responses against AAV capsid and transgene products.

“Zhu et al., using murine model and in vitro cultures of human peripheral blood mononuclear cells (PBMCs) demonstrated that pDCs but not conventional DCs (cDCs) or macrophages respond to the AAV genome through TLR9-MyD88 pathway leading to production of IFN I, which promotes adaptive immune responses against AAV capsid, as well as AAV encoded transgene products.”

• cites [22] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

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.”

• cites [23] sourceNot checked yet

  Not checked in the free run.

• cites [24] sourceNot checked yet

  Not checked in the free run.

• cites [25] sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S4 (based).

Plasmacytoid dendritic cells can promote CD8+ T cell responses independently of TLR9 by producing IL-1 cytokines that activate IL-1R1>MyD88 signaling.

“Alternatively, pDCs may also promote CD8 + T cell responses in a TLR9-independent manner by the production of IL-1 cytokines, thereby activating the IL-1R1>MyD88 signaling pathway.”

May combine two claims.

AAV ITRs have intrinsic low-level transcriptional activity and therefore could potentially generate complementary RNA strands.

“AAV ITRs are known to have intrinsic low-level transcriptional activity, 52,53 therefore could potentially generate complementary strands of RNAs.”

• cites [52] sourceNot checked yet

  Not checked in the free run.

• cites [53] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Transient inhibition of MDA5 and its downstream adaptor MAVS in HeLa cells increased transgene expression, presumably by preventing IFN-β expression.

“Transient inhibition of MDA5 and its downstream adaptor mitochondrial antiviral signaling (MAVS) in HeLa cells was able to increase the transgene expression, presumably by preventing IFN-b expression.”

• cites [55] sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Whether dsRNA expression plays a significant role in AAV gene transfer is not yet clear, and no links to adaptive immune responses or tissue inflammation have been established.

“Whether dsRNA expression plays a significant role in AAV gene transfer is not yet clear, and no links to adaptive immune responses or tissue inflammation have been established.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Complement activation in tissue may raise interstitial fluid and lead to organ dysfunction, particularly in the heart.

“Tissue level complement activation may increase interstitial fluid and lead to organ dysfunction, especially in the heart.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

C3b binding to C3 convertase forms C5 convertase, which cleaves C5 into C5a and C5b.

“Association of C3b to C3 convertase leads to the formation of C5 convertase (C4b2a3b for classical and lectin pathways; C3bBb3b for the alternative pathway), which cleaves C5 to anaphylatoxin C5a and opsonin C5b.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Several other studies showed no effect of STING deficiency on B or T cell responses.

“Several other studies showed no effect of STING deficiency on B or T cell responses.”

• cites [26] sourceNot checked yet

  Not checked in the free run.

• cites [35]Not checked yet

  Not checked in the free run.

May combine two claims.

In humans, the classical pathway is critical for initiating complement activation and associated pathologies after high-dose systemic AAV, while the alternative pathway may serve as an amplification loop.

“Based on these findings, the authors proposed that in humans, the classical pathway is critical for the initiation of complement activation and the occurrence of associated pathologies/toxicities following high-dose systemic delivery of AAV, whereas the alternative pathway may serve as an amplification loop.”

• cites [89] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Clinical symptoms possibly related to complement activation include thrombotic microangiopathy, acute kidney injury involving atypical hemolytic uremic syndrome, thrombocytopenia, elevated transaminases, and endothelial injury.

“Clinical symptoms that may be related to complement activation included thrombotic microangiopathy (TMA), acute kidney injury involving atypical hemolytic uremic syndrome, thrombocytopenia, elevated transaminases, and endothelial injury.”

May combine two claims.

Smith et al. found that preexisting anti-AAV antibody titers of at least 1:100 efficiently activated complement and increased proinflammatory cytokine secretion and cDC activation, independently of whether vector genomes were empty or full.

“Smith et al. found that higher titers of preexisting anti-AAV antibodies ( ‡1:100) were highly efficient in activation of the complement system and increased secretion of proinflammatory cytokines and activation of cDCs, independent of the vector genome (i.e., empty vs. full particles).86”

• cites [86] sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

C5b recruits C6 through C9 to form the lytic membrane attack complex on the pathogen surface.

“C5b then recruits other complement proteins, C6-C9, to form the lytic membrane attack complex on the surface of the pathogen.”

May combine two claims.

AAV capsid lacks cofactor activity for factor I-mediated conversion of C3b to iC3b and instead binds complement regulatory protein factor H.

“AAV capsid lacked cofactor activity for factor I-mediated degradation of C3b to iC3b and instead also bound complement regulatory protein, factor H.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

All three complement activation pathways converge at the C3 convertase step, which then cleaves C3 into C3a and C3b.

“Despite different means of activation, all three pathways converge at the C3 convertase stage (C4b2a for classical and lectin pathways; C3bBb for the alternative pathway), which subsequently catalyzes C3 to anaphylatoxin C3a and opsonin C3b.”

• cites [78] sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Zaiss et al. first reported in 2008 that AAV can interact with complement proteins.

“In 2008, Zaiss et al. first reported that AAV could interact with complement proteins.82”

• cites [82] sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Complement activity is tightly controlled by soluble and surface-bound regulatory proteins to avoid unnecessary damage to host tissues and organs.

“Because the complement cascade leads to generation of highly potent inflammatory molecules, its activity is tightly regulated by a variety of soluble and surface-bound regulatory proteins, thus preventing unnecessary damage to host tissues and organs.80,81”

• cites [80] sourceNot checked yet

  Not checked in the free run.

• cites [81] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Complement may mediate AAV vector uptake and trigger pro-inflammatory cytokines in primary murine macrophages and a human monocytic cell line.

“The authors found that complement may mediate the uptake of AAV vector and trigger pro-inflammatory cytokines in primary murine macrophages and a human monocytic cell line.82”

• cites [82] sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

C3a and C5a are pro-inflammatory anaphylatoxins that drive chemotaxis and activation of granulocytes, mast cells, and macrophages and open endothelial tight junctions, causing tissue edema.

“Anaphylatoxins (C3a, C5a) generated during complement cascade are generally pro-inflammatory and are responsible for chemotaxis and activation of granulocytes, mast cells, and macrophages to the site of infection 79 through opening of endothelial tight junctions leading to tissue edema.”

• cites [79] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Factor H may restrain alternative pathway complement activation but can enhance B cell activation through C3d generation and engagement of complement receptors on B cells and dendritic cells.

“The latter may limit complement activation through the alternative pathway but enhances B cell activation through generation of C3d, resulting in engagement of complement receptors on B cells and DCs.”

May combine two claims.

West et al. observed complement activation when serum had higher preexisting anti-AAV IgG1 antibodies, and this activation was significantly reduced after immunoglobulin depletion.

“Similarly, West et al. also observed activation of the complement system when serum samples had higher levels of preexisting anti-AAV antibodies (IgG1), which were significantly reduced when immunoglobulins were depleted.87”

• cites [87] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Emerging data from Verdera et al. demonstrate that RNA sensing through MAVS may drive AAV-induced antiviral responses in certain cell types, as MAVS inhibition in human iPSC-derived astrocytes comprehensively reduced interferon-stimulated gene expression.

“Furthermore, emerging data from Verdera et al. demonstrate that RNA sensing through MAVS may drive AAV-induced antiviral responses in certain cell types, as inhibition of MAVS in cultures of human induced pluripotent stem cell-derived astrocytes comprehensively reduced expression of interferon stimulated genes.”

• cites [48] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Activation of the cGAS-STING pathway in these CNS cultures was downstream of p53 upregulation, and STING inhibition with H151 abrogated the pro-inflammatory response.

“Interestingly, authors found that activation of cGAS-STING pathway is downstream of p53 (a tumor suppressor gene) upregulation, and inhibition of STING with an antagonist (H151) abrogated the pro-inflammatory response.”

• cites [48] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Patients treated with rituximab and sirolimus in addition to corticosteroids had no IgM or IgG elevations and lacked activated complement proteins.

“Patients treated with rituximab and sirolimus in addition to corticosteroid had no elevations of IgM or IgG and lacked activated complement proteins.”

• cites [89] sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

The monoclonal antibody eculizumab, a C5 inhibitor, is currently used in attempts to reduce complement-related immunotoxicities.

“Currently, monoclonal antibody eculizumab (an inhibitor of C5) is being used in an attempt to lessen complementrelated immunotoxicities.13,95”

• cites [13] sourceNot checked yet

  Not checked in the free run.

• cites [95] sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Activation of the cGAS-STING pathway may impact gene editing applications that use AAV vectors to deliver DNA templates, such as ex vivo editing of hematopoietic cells.

“However, activation of this pathway may impact gene editing applications that use AAV vectors to deliver DNA templates, for example, in ex vivo editing of hematopoietic cells.”

Probably not a substantial claim: may not pass S3 (load-bearing).

An alternative protocol targeting CD20 and the B cell survival factor BAFF may also be useful.

“An alternative protocol targeting CD20 and the B cell survival factor BAFF may also be useful in this regard.94”

• cites [94] sourceNot checked yet

  Not checked in the free run.

May combine two claims.

C3 binds directly to AAV capsid, but later work suggests that AAV complement activation is antibody dependent and proceeds through the classical pathway.

“Although C3 was found to bind directly to AAV capsid, further studies suggested that complement activation by AAV is antibody dependent and follows the classical pathway.”

May combine two claims.

Complement activation after AAV administration was not seen in clinical trials until very high systemic doses were used, which may now be known to trigger life-threatening immunotoxicities.

“Complement activation following AAV administration had not been observed in clinical trials until very high doses were used for systemic delivery, which we now know may trigger life-threatening immunotoxicities.12,13,88,[90][91][92]”

• cites [12] sourceNot checked yet

  Not checked in the free run.

• cites [13] sourceNot checked yet

  Not checked in the free run.

• cites [88]Not checked yet

  Not checked in the free run.

May combine two claims.

IdeS does not affect IgM, which can activate complement and forms within days after AAV infusion.

“However, IdeS does not impact IgM, which is equally capable of activating complement system and readily forms within days after AAV infusion.”

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

Most capsids can activate complement in in vitro assays with antibodies, but differences in biodistribution and half-life could possibly matter in vivo.

“While most capsids are able to activate complement in in vitro assays in the presence of antibodies, differences in their biodistribution and half-life in circulation could possibly make a difference in vivo.”

May combine two claims.

TLR9>MyD88 and IL-1R1>MyD88 signaling is critical for cellular responses to the AAV-encoded transgene product in liver.

“TLR9>MyD88 and IL-1R1>MyD88 mediated signaling is critical for cellular responses to the AAV-encoded transgene product in liver.”

May combine two claims.

Some internalized AAV capsids are degraded into peptides in endosomes, while those escaping endosomes are degraded in the cytoplasm by proteasomes.

“Some of the internalized AAV capsids are proteolytically degraded into smaller peptides in endosomes, whereas some of those that manage to escape endosomes are degraded in the cytoplasm (by proteasomes).”

May combine two claims.

Further complement analysis in primates indicated alternative pathway activation, with elevated Bb and SC5b-9 but not C4a.

“Further analysis of complement proteins indicated alternative pathway activation, as elevated levels of Bb (complement protein associated with alternative pathway) and terminal complement complex SC5b-9 but not C4a were observed.”

• cites [96] sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The signaling requirements for reactivating memory responses are usually lower than those for primary responses.

“For example, the signaling requirements for reactivation of memory responses are usually lower than for primary responses.”

May combine two claims.

AAV vectors carry weaker inflammatory signals than many other viruses or vector systems, but they still elicit innate responses that can substantially affect gene therapy.

“Although AAV vectors contain weaker inflammatory signals than many other viruses or vector systems, they nonetheless elicit innate responses that can have a significant impact on gene therapy.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

C1 inhibitor is being evaluated in clinical trials to block the classical pathway, while a C3 inhibitor could theoretically block all three complement pathways.

“For instance, C1 inhibitor for blocking of classical pathway is being evaluated in clinical trial, whereas C3 inhibitor theoretically could block the activation of all three complement pathways.”

May combine two claims.

Antigen-primed and activated T cells leave the liver to survey other organs and can be detected in the circulation.

“Antigen primed and activated T cells egress from the liver to survey other organs and can be detected in the circulation (E).”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Although these pathways use different C3 convertases, they follow the same subsequent steps to form the membrane attack complex.

“Although these pathways utilize different C3 convertases, they follow the same subsequent steps leading up to the formation of the membrane attack complex.”

Probably not a substantial claim: may not pass S4 (based).

AAV inverted terminal repeats have inherent transcriptional activity that could produce double-stranded RNA.

“AAV ITRs have inherent transcriptional activity, which could lead to the formation of dsRNA.”

May combine two claims.

The classical complement pathway is activated when C1q recognizes AAV-antibody complexes, while binding of hydrolyzed C3b on AAV activates the alternative pathway.

“The classical pathway of complement is activated upon recognition of AAV-antibody complexes by C1q, whereas binding of hydrolyzed C3b on AAV activates the alternative pathway.”

May combine two claims.

This transient antiviral environment depends on Kupffer cells and involves infiltration of innate cells including neutrophils and natural killer cells.

“This transient antiviral environment is dependent on KCs and involves infiltration of innate cells, including neutrophils and NK cells (B).”

May combine two claims.

This process facilitates AAV uptake, processing, and presentation of AAV-derived antigens to T cells, leading to an effective humoral and cellular response to AAV.

“This process facilitates AAV uptake, processing, and presentation of AAV-derived antigens to T cells leading to the generation of an effective humoral and cellular response to AAV.”

The quote only partly states this claim.

Cytoplasmic double-stranded RNA sensors such as RIG-I and MDA5 may recognize AAV-derived RNA and drive IFN-I secretion.

“Cytoplasmic dsRNA sensors such as RIG-I and MDA5 may recognize and drive the secretion of IFN-I.”

May combine two claims.

A deeper understanding of innate immune mechanisms against AAV has enabled engineering of less immunogenic vectors and better immune suppression protocols.

“A deeper understanding of innate immune response mechanisms against AAV has led to the engineering of vectors with reduced immunogenicity and the development of superior immune suppression protocols.”

May combine two claims.

TLR2 recognizes AAV capsid, while TLR9 detects unmethylated CpG motifs in the AAV genome.

“TLR2 mediates recognition of AAV capsid, whereas TLR9 recognizes unmethylated CpG motifs in the AAV genome.”

May combine two claims.

Vector-related severe adverse events in primates included severe thrombocytopenia, transaminase elevations, edema, acute endothelial injury, cholestatic effects, and hepatotoxicity, with similarities to clinical studies in DMD, spinal muscular atrophy, and X-linked myotubular myopathy.

“Vector-related severe adverse events included severe thrombocytopenia, transaminase elevations, edema, acute endothelial injury, cholestatic, and hepatotoxicity, among others, and showed some similarities to the ones observed in clinical studies of DMD, spinal muscular atrophy, and Xlinked myotubular myopathy.12,13,17,97,98”

• cites [12] sourceNot checked yet

  Not checked in the free run.

• cites [13] sourceNot checked yet

  Not checked in the free run.

• cites [17] sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

Innate immunity is connected to adaptive responses and to immunotoxicities.

“Innate immunity links to adaptive responses and to immunotoxicities.”

May combine two claims.

The early time point of these primate toxicities would be during IgM formation and may therefore not reflect classical pathway activation.

“This time point would be early during IgM formation and may therefore not reflect classical pathway activation.”

May combine two claims.

IL-1-producing plasmacytoid dendritic cells begin to infiltrate the liver 3 days after AAV administration and peak around day 7.

“IL-1 producing pDCs begin to infiltrate the liver 3 days after AAV administration and peak around day 7.”

May combine two claims.

These events lead to neutrophil extracellular trap formation and platelet aggregation.

“These events lead to the formation of neutrophil extracellular traps and platelet aggregation (C).”