Early Changes in Liver Transcriptomic Profiles Following Adeno-Associated Viral Gene Therapy in the Severe Hemophilia A Dog Model
Bibliographic record
Abstract
Introduction Recombinant adeno-associated virus vectors (rAAV) are a major gene therapy platform for treatment of monogenic disorders, including hemophilia A. Valoctocogene roxaparvavec (AAV5-HLP-hFVIII-SQ) is an AAV5 vector delivering B-domain-deleted (BDD) human FVIII transgene controlled by a hybrid liver-selective promoter. In a phase 3 trial, a single treatment of AAV5-HLP-hFVIII-SQ (6e13 vg/kg) provided therapeutic expression of FVIII and bleeding control in adult severe hemophilia A patients. However, the mechanistic basis of transaminitis, variability and durability observed in clinical trial participants are not clear. Understanding the molecular changes in the liver are critical to identify appropriate immune modulatory strategies for safety, efficacy, and long-term durability of AAV gene therapy. The aim is to investigate liver gene expression profiles before and after AAV5-HLP-canine-BDD-FVIII (cFVIII) administration in a severe hemophilia A dog model. Method Nine severe hemophilia A dogs received one of three vectors (non-codon-optimized AAV5-HLP-cFVIII-SQ, codon-optimized AAV5-HLP-cFVIII-SQ and AAV5-HLP-cFVIII-V3 at 6.0e13 – 2.0e14 vg/kg). Liver biopsies were collected at baseline and 3 months after vector administration. FVIII activity (FVIII:C) was measured by one-stage FVIII (OSA) assay using a pooled normal canine plasma standard. Liver cFVIII DNA and RNA levels were quantified using ddPCR. Transcriptomic profiling was performed by RNA-seq followed by pathway enrichment analysis. Cellular immune response in the peripheral blood mononuclear cells (PBMC) was evaluated using an IFN-γ ELISPOT assay. Results Dose-relatedFVIII expression was observed in dogs treated with the codon-optimized vectors, with significant correlation between liver vector DNA and circulating FVIII:C ([ Fig. 1 ]). Dogs treated with codon-optimized cFVIII vectors demonstrated enhanced transgene expression compared to non-codon-optimized vector. No IFN-γ response was detected in PBMC. Transcriptomic profiling of liver biopsies demonstrated enrichment of integrin pathways, immunological gene signatures for B cells and plasmacytoid dendritic cells (pDCs), and common dendritic cells at 3 months compared to baseline ([ Fig. 2 ]). Expression of inflammatory cytokines involved in NK-cell and T-cell activation were also enriched. Fig. 1 AAV5-cFVIII expression and activity in severe hemophilia A dogs at 3 months ; ( a ) AAV5-cFVIII mRNA transcript levels, and ( b ) correlation between liver vector DNA and OSA FVIII:C, one-stage FVIII clotting assay (canine normal pooled standard) with a lower limit of detection of 2%. co, codon optimized; nco, non-codon optimized; SQ and V3, B-domain-deleted FVIII variants. Fig. 2 Gene set enrichment analysis comparing AAV5-HLP-cFVIII vectors administered to hemophilia A dogs at 3 months and baseline (treatment vs control). A normalized gene set enrichment score (NES) determines whether a gene set is positively regulated or negatively regulated and an NES with Benjamini-Hochberg adjusted P value<0.05 was considered statistically significant. FDR, False discovery rate. Conclusion Our data suggests that mild activation of B cells, dendritic cells, NK-cell, and T-cells with an inflammatory cytokine response occurred in the liver of AAV5-HLP-cFVIII treated dogs 3 months post gene transfer; albeit without transaminitis. Transcriptomic profiling of PBMCs is ongoing to compare to liver profiles to better understand kinetics of immune responses to rAAV. Publication History Article published online: 26 February 2024 © 2024. Thieme. All rights reserved. Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".