Innate and Adaptive Immune Responses to Adeno-Associated Viral Gene Therapy in the Severe Hemophilia A Dog Model

P. Batty¹, A. Mo², M. Ashrafali³, B. Yates³, A. Gonzalez³, B. Handyside³, L. Harpell², D. Hurlbut², A. Menard⁴, A. Pender², L. Razon³, C. Sihn³, A. Winterborn², S. Fong³, D. Lillicrap⁵

¹University College London, London, United Kingdom, ²Queen's University, Canada, Kingston, Ontario, Canada, ³BioMarin Pharmaceutical, USA, Novato, California, United States, ⁴Kingston Health Sciences Center, Canada, Kingston, Ontario, Canada, ⁵Department of Pathology and Molecular Medicine, Queen’s University, Kingston, Ontario, Canada

Abstract Number: OC 01.5

Meeting: ISTH 2022 Congress

Theme: Hemophilia and Rare Bleeding Disorders » Hemophilia Gene Therapy

Background: Adeno-associated viral (AAV) gene therapy is under investigation for the treatment of severe hemophilia. The predominant adverse event in clinical trials has been elevation of alanine aminotransferase (ALT) levels. Although capsid directed cytotoxic T-cell responses have been reported, the underlying mechanism(s) remain unclear.

Aims: To evaluate the early immune response to AAV5-cFVIII in the severe hemophilia dog model.

Methods: Severe hemophilia A dogs were treated using one of three AAV5-B-domain deleted canine Factor VIII constructs with a hybrid liver promoter (AAV5-HLP-cFVIII, Table 1). Capsid immune responses were evaluated using total antibody and ELISPOT assays. Serum innate immune responses were evaluated using a cytokine array (n=16) and complement function. Vector genome (VG) copies and cFVIII mRNA were quantified in liver biopsies at baseline and 3-months post-treatment using ddPCR.

Results: Ten male hemophilia dogs (median = 4.3 years, 0.8 – 9.0 years) received a single AAV5-HLP-cFVIII infusion (dose = 6.0e13 – 2.0e14 vg/kg). FVIII expression was seen for dogs treated with the codon-optimized constructs (n=7). Dose response was seen at 3 months post-treatment, with higher one-stage FVIII:C in the high-dose (2e14 vg/kg, mean=15.6±0.3 IU/dL) compared to the low-dose cohort (6-8e13 vg/kg, mean=6.2±1.0 IU/dL). Liver biopsies at 3-months demonstrated AAV5-HLP-cFVIII in all animals, with correlation between VG and cFVIII mRNA copies (r=0.8, p=0.01). Anti-AAV5 capsid antibodies were detected starting day 7 post-treatment. No new FVIII inhibitors were detected. Dogs displayed variable serum cytokine profiles, with no change in mean levels 24-hours post-dosing. Higher baseline and post-dose levels of TNF-alpha, IL2, IL6, IL7, IL15 and IL18 were observed in the dog with a FVIII inhibitor history (MEM2018). No capsid specific T-cell responses or complement activation was detected.

Conclusion(s): FVIII expression was seen using co-AAV5-HLP-cFVIII vectors in the hemophilia dog model. No blood biomarkers of innate immune activation were detected, with further liver studies ongoing.

Table

Table 1: Outcomes following AAV5-HLP-cFVIII in the severe hemophilia A dog model. HLP = Hybrid liver promoter. nco = non-codon optimized. co = codon optimized. dg = diploid genome. OSA = One stage FVIII:C -canine normal pooled standard-. Follow up as of January 17th 2021 or study end-point. * History of FVIII inhibitor. WBCT = Whole blood clot time -normal: 4-6 minutes-. n/a = not available.

To cite this abstract in AMA style:

Batty P, Mo A, Ashrafali M, Yates B, Gonzalez A, Handyside B, Harpell L, Hurlbut D, Menard A, Pender A, Razon L, Sihn C, Winterborn A, Fong S, Lillicrap D. Innate and Adaptive Immune Responses to Adeno-Associated Viral Gene Therapy in the Severe Hemophilia A Dog Model [abstract]. https://abstracts.isth.org/abstract/innate-and-adaptive-immune-responses-to-adeno-associated-viral-gene-therapy-in-the-severe-hemophilia-a-dog-model/. Accessed September 29, 2023.

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