Abstract Number: PB1087
Meeting: ISTH 2020 Congress
Background: Gene therapy using adeno-associated viral (AAV) vectors has demonstrated durable Factor VIII (FVIII) expression in patients and animal models of severe hemophilia A. The mechanisms mediating long-term expression and capsid immune responses are not known.
Aims: To evaluate the site of expression, vector DNA forms and capsid immunogenicity to AAV-FVIII in a hemophilia dog model.
Methods: Hemophilia A dogs received a single canine-FVIII AAV vector infusion (AAV-BDD-cFVIII, AAV2=4, AAV6=3 and AAV8=1). FVIII activity (FVIII:C) was measured using pooled canine plasma as a standard. AAV-BDD-cFVIII vector genomes and BDD-cFVIII mRNA were quantified by qPCR and qRT-PCR. Circular full-length AAV episomes were quantified using droplet-digital PCR. Anti-AAV capsid neutralizing antibody (nAb) titres were measured using a cell-based in-vitro transduction inhibition assay.
Results: Eight dogs received a median dose of 1.25×1013 vg/kg AAV-BDD-cFVIII by portal vein infusion (Table 1). After 10.8 years (8.2-12.0 years), persistent FVIII:C (median one-stage=12.7%, chromogenic=7.2%) was seen in all responding dogs (n=6), with improvement in bleeding phenotype. AAV-BDD-cFVIII DNA was detected in the liver of all dogs (median=30.5 vg/ng), with lower levels in the spleen in 4 dogs (median=1.0 vg/ng). cFVIII mRNA was only detected in the liver of responding dogs (median=7.3 copies/ng). Hepatic cFVIII mRNA copies were significantly associated with FVIII:C. Full-length linear and circular AAV-BDD-cFVIII was detected in the liver of all animals. Anti-AAV capsid nAbs were detected throughout the study: mean earliest available peak post-treatment titre 6485. Most samples demonstrated limited cross-reactivity to other capsids. Although a decline in nAb titre was seen with time (coefficient= -314, p< 0.001), substantial nAbs remained detectable (mean final titre: 809).
Conclusions: Persistent liver-derived FVIII expression was seen 10 years after a single AAV-BDD-cFVIII infusion, with detectable full-length linear and circular AAV-FVIII forms. Although anti-capsid humoral immune responses decrease with time, these remain at levels potentially preventing redosing with the same capsid.
|ID (Sex)||AAV Serotype (dose vg/kg)||Final Chromogenic FVIII:C (years)||Liver BDD-cFVIII vg/ng DNA||Spleen BDD-cFVIII vg/ng DNA||Liver BDD-cFVIII copies/ng mRNA||Spleen BDD-cFVIII copies/ng mRNA||Liver Full-length Circular AAV-FVIII vg/diploid genome||Peak AAV nAb titre (years)||Late AAV nAb titre (years)|
|EL (F)||2 (6.0e12)||n/a (8.2)||8.9||0.3||6.2||nd||0.014||22068 (3.6)||n/a|
|VC (M)||2 (1.5e13)||2.9% (11.0)||120.4||nd||8.3||nd||0.096||6110 (2.4)||220 (11.0)|
|ANG (M)||2 (1.5e13)||<1% (11.4)||10.7||1.0||nd||nd||0.007||5631 (7.9)||1445 (11.5)|
|JU (M)||2 (2.7e13)||7.2% (11.9)||50.2||1.0||15.9||nd||0.003||5092 (3.2)||1864 (11.9)|
|ALX (F)||6 (1.0e13)||8.6% (10.5)||36.0||nd||26.4||nd||0.089||4130 (1.5)||293 (10.5)|
|MZ (M)||6 (1.0e13)||7.9% (10.1)||35.1||nd||5.9||nd||0.054||4805 (1.2)||634 (10.1)|
|MG (F)||6 (1.7e13)||<1% (11.3)||2.8||nd||nd||nd||0.002||761 (8.0)||647 (11.5)|
|FLO (F)||8 (1.0e13)||1.8% (9.8)||25.9||6.8||4.4||nd||0.018||3280 (1.0)||559 (10.5)|
[Table 1: FVIII expression, vector genomes and capsid immune response post AAV-FVIII. nd=not detected]
To cite this abstract in AMA style:Batty P, Sihn CR, Ishida J, Mo AM, Yates B, Brown C, Harpell L, Pender A, Russell C, Sardo Infirri S, Torres R, Winterborn A, Fong S, Lillicrap D. Long-Term Vector Genome Outcomes and Immunogenicity of AAV FVIII Gene Transfer in the Hemophilia A Dog Model [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/long-term-vector-genome-outcomes-and-immunogenicity-of-aav-fviii-gene-transfer-in-the-hemophilia-a-dog-model/. Accessed November 30, 2023.
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