Self-Complementary AAV Vectors Cause a Substantially Heightened TLR9-Dependent Innate Immune Response In the Liver
Notice bibliographique
Résumé
Abstract Abstract 252 Successful gene therapy for hemophilia requires therapeutic expression in the absence of destructive immune responses. Clinical trials have revealed that adaptive immune responses eliminated therapeutic levels of factor IX (F.IX) following high-dose hepatic in vivo gene transfer using single stranded (ss) AAV serotype 2 vector. More efficient AAV vectors have been developed using AAV2 with Y-F capsid mutations, alternate serotypes such as AAV8, and self-complimentary (sc) rAAV vectors. Innate immune responses to AAV in the liver are thought to be weak and highly transient. Our study sought to investigate whether variations in the capsid or genome will alter the immune profile. AAV vectors were delivered into the portal circulation of C3H/OuJ mice at 1011 vector genomes per mouse. We tested ssAAV-2, ssAAV2-triple Y-F mutant, ssAAV-8 vectors as well as scAAV-2 and scAAV-8 vectors, all expressing the hF.IX transgene. Quantitative RT-PCR array showed a mild transient up-regulation of MyD88, TLR-9, TNF-α, MCP-1, IP-10 and IFN-α/β within 2 hrs, which subsided by 6 hrs following delivery of ssAAV vectors, regardless of the capsid sequence/efficiency of hepatocyte transduction. In contrast, scAAV-2 and scAAV-8 vectors induced 4- to 8-fold increases in TLR-2, TLR-9, MyD88, TNF-α, MCP-1, IP-10, and IFN-α/β expression when compared to the ssAAV vectors. In addition, scAAV vectors induced expression of pro-inflammatory cytokines and chemokines not detected for ssAAV: IL-6, IL-12α, MIP-1, and RANTES. Paralleling this data was a 4-fold increase in local protein levels of IL-6, TNF-α, and MCP-1, and a systemic increase in IL-6. None of the vectors activated TLR-4, indicating that these effects cannot be attributed to LPS contamination. Several other innate response genes showed no differences in expression compared to PBS injected mice, which included IL-1α, IL-1β, KC, TLR-1, and TLR-3 to -8. The heightened innate response to scAAV vectors was further characterized by a 5-fold increase in macrophage and neutrophil infiltrates in liver sections, illustrating the potential for hepatotoxicity. Innate responses were absent in TLR-9 deficient C57BL/6 mice and in wild-type mice that had received an oligonucleotide ligand inhibitory to TLR-9. Compared to wild-type mice, hFIX levels were increased 2-fold in TLR-9 deficient mice following scAAV vector delivery and antibody formation against capsid was delayed. Additionally, in vitro studies with a TLR-9 reporter cell line showed significantly increased TLR-9 signaling for scAAV2 compared to ssAAV vectors, independent of the transgene/expression cassette. Neither vector activated the inflammasome. Further in vivo studies showed that the innate response to scAAV was, with the exception of TNF-a expression, Kupffer cell dependent, likely owing to the importance of these cells to sequester viral particles in the liver. Our results have several implications. Gene therapy applications based on administration of high-dose scAAV may be limited by immunotoxicities, which, however, can be prevented by inhibition of TLR-9 signaling. Of general importance, changing the genome configuration of a virus from single- to double-stranded DNA increased sensing by the endosomal receptor TLR-9, thereby enhancing innate immunity. Disclosures: Herzog: Genzyme Corp: Patents & Royalties.
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».