Abstract Number: PB1088
Meeting: ISTH 2020 Congress
Theme: Hemophilia and Rare Bleeding Disorders » Hemophilia Gene Therapy
Background: Liver-derived Factor VIII (FVIII) expression has been described in the canine hemophilia A model for more than 10 years following adeno-associated viral (AAV) gene therapy. Improved understanding of AAV integration may provide insights into the long-term safety of AAV based gene therapy.
Aims: To evaluate frequency and locations of AAV-FVIII integration sites in a canine hemophilia model.
Methods: Eight severe hemophilia A dogs received a portal vein infusion of canine-FVIII-AAV vector (AAV-BDD-cFVIII). Genomic DNA was isolated from post-mortem liver samples. AAV-BDD-cFVIII vector genomes (vg/ng) were quantified by qPCR. Vector integration sites (IS) were analysed by linear amplification-mediated (LAM)-PCR and target-enrichment sequencing (TES) coupled to Illumina MiSeq sequencing and customised bioinformatical analysis.
Results: AAV-BDD-cFVIII DNA was detected in the liver of all dogs, 10.8 years after a single vector infusion (Table 1). LAM-PCR and TES analyses demonstrated overlapping IS and low integration frequencies in all animals: median=9.55e-4 IS/cell by TES and 4.50e-04 IS/cell by LAM-PCR. IS number correlated with vg/ng copies, but not with AAV dose or gender. Most integration events (93.8%) occurred in intergenic regions of the dog genome (upstream=49.0%, downstream=44.2%, in-gene=6.3% and unannotated=0.6%). The most frequent in-gene integration events occurred within the F8 (all animals, intronic=59, exonic=143) and albumin loci (7 animals, intronic=22, exonic=1). All other exonic insertions (n=8) were random appearing single gene events. Analysis of the top 10 IS in each sample, revealed IS in proximity to 81 genes, with the most frequent recurrent sites being MIR1296 (downstream=18), KCNIP2 (downstream=16), ABCB1 (upstream=13), CLIC2 (upstream=12), F8 (in-gene=6) and MET (upstream=5). Despite integration events occurring in all animals, no tumors were found within the liver at post-mortem.
Conclusions: Low hepatic AAV vector integration frequencies, at rates lower than the spontaneous natural human mutation rate, with some apparent recurrent loci were detected 10 years after a single dose of AAV-FVIII.
| ID | Dose Capsid cFVIII:C | Mean vg/ng (vg/cell) | TES Mean IS/vg | TES Mean IS/cell | TES Mean IS | Mean Upstream IS (%) | Mean In-gene IS (%) | Mean Downstream IS (%) | Mean Unannotated IS (%) |
| VC | 1.5e13 AAV2 2.9% | 120.4 (7.94e-1) | 4.38e-3 | 3.48e-3 | 665.5 | 315 (47.3%) | 47 (7.1%) | 300.5 (45.2%) | 3 (0.5%) |
| JU | 2.7e13 AAV2 7.2% | 50.2 (3.31e-1) | 5.48e-3 | 1.82e-3 | 542 | 225 (41.5%) | 40.5 (7.5%) | 273 (50.4%) | 3.5 (0.6%) |
| ALX | 1.0e13 AAV6 8.6% | 36.0 (2.38e-1) | 3.32e-3 | 7.89e-4 | 336.5 | 162.5 (48.3%) | 16.5 (4.9%) | 155.5 (46.2%) | 2 (0.6%) |
| MZ | 1.0e13 AAV6 7.9% | 35.1 (2.32e-1) | 3.46e-3 | 8.02e-4 | 298 | 176.5 (59.2%) | 17.5 (5.9%) | 100.5 (33.7%) | 3.5 (1.2%) |
| FLO | 1.0e13 AAV8 1.8% | 25.9 (1.71e-1) | 7.00e-3 | 1.19e-3 | 387.5 | 204.5 (52.8%) | 19 (4.9%) | 162 (41.8%) | 2 (0.5%) |
| ELI | 6.0e12 AAV2 n/a | 8.9 (5.87e-2) | 1.51e-2 | 8.88e-4 | 382.5 | 187 (48.9%) | 22.5 (5.9%) | 171.5 (44.8%) | 1.5 (0.4%) |
| ANG | 1.5e13 AAV2 <1% (nr) | 10.7 (7.03e-2) | 1.46e-2 | 1.02e-3 | 184 | 98.5 (53.5%) | 10.5 (5.7%) | 74 (40.2%) | 1 (0.5%) |
| MG | 1.7e13 AAV6 <1% (nr) | 2.8 (1.82e-2) | 8.91e-3 | 1.62e-4 | 102 | 52 (51.0%) | 6.5 (6.4%) | 43 (42.2%) | 0.5 (0.5%) |
[Table 1: Targeted Enrichment Sequencing of Integration Events in a Canine Hemophilia. nr=no response]
To cite this abstract in AMA style:
Batty P, Fong S, Franco M, Gil-Farina I, Mo AM, Harpell L, Hough C, Hurlbut D, Pender A, Sardo Infirri S, Winterborn A, Schmidt M, Lillicrap D. Frequency, Location and Nature of AAV Vector Insertions After Long-Term Follow up of FVIII Transgene Delivery in a Hemophilia A Dog Model [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/frequency-location-and-nature-of-aav-vector-insertions-after-long-term-follow-up-of-fviii-transgene-delivery-in-a-hemophilia-a-dog-model/. Accessed November 29, 2023.« Back to ISTH 2020 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/frequency-location-and-nature-of-aav-vector-insertions-after-long-term-follow-up-of-fviii-transgene-delivery-in-a-hemophilia-a-dog-model/