PB0228 - The effect of gut microbial dysbiosis on the efficacy, variability, and durability of liver-directed AAV-mediated gene therapy

Sunday, June 23, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

FB

Fateme Babaha

PhD Student
Department of Pathology and Molecular Medicine, Queen's University, Kingston, Canada
Kingston, Ontario, Canada

Co-Author(s)

EB

Erin Burnett

Veterinary Technician & Registered Laboratory Animal Technician (VT RLAT)
Department of Pathology and Molecular Medicine, Queens University
Kingston, Ontario, Canada
Maria Rita Peralta, MD

Haemostasis&Thrombosis Consultant
St Thomas Hospital & Royal Brompton Hospital, London, UK
LONDON, England, United Kingdom
AM

Aomei Mo

Research Technologist
Department of Pathology and Molecular Medicine, Queen's University, Kingston, Canada
Kingston, Ontario, Canada
Paul Batty, MBBS PhD

Associate Professor
Cancer Institute, University College London, London, UK
London, England, United Kingdom
David Lillicrap, MD

Professor Department of Pathology and Molecular Medicine
Molecular Hemostasis Research Group, Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada.
Kingston, Ontario, Canada

Background: Alterations in the gut bacterial composition, or dysbiosis, are recognized to alter the immune state of the liver. How these changes influence liver-directed gene therapy is unknown.

Aims: To evaluate the pattern of transgene expression and immune response in mice following induction of gut microbial dysbiosis.

Methods: Dysbiosis was induced using a 10-day course of 4 antibiotics (ABX). Cecal content was collected for microbiome analysis. 25 wildtype C57BL/6 mice, with and without ABX therapy, received a reporter AAV5 vector expressing a human Alpha1 anti-trypsin transgene (AAV5-hA1AT) (dose=6e13 vg/kg). F8 knock-out (F8KO) mice, with or without ABX induced dysbiosis (n=17), received a codon-optimized AAV5-canine FVIII (AAV5-cFVIII-SQ) vector (dose=6e13 vg/kg). Serial blood collection to measure A1AT and FVIII:C (chromogenic) expression was performed for 5 months.

Results: 16s rRNA sequencing showed that dysbiosis of gut microbiota was achieved (Figure 1). In normal mice, starting from 2 weeks post-delivery, A1AT expression was seen which peaked at 3 months (48.2- 63.2 ng/ml) followed by a decline at 4 months (Figure 2A). In F8KO/ABX-treated mice, peak FVIII:C expression was seen 2-weeks post-delivery (FVIII:C 63%), with a fall (mean FVIII:C = 20%) at 2 months, which remained stable for the next 3 months. In non-ABX treated mice, FVIII:C expression started 2-weeks post-delivery, peaked at 2 months (FVIII:C 46%), then dropped and stabilized (mean FVIII:C = 10%) for the remainder of the study (Figure 2B). For both vectors, no significant difference in transgene expression was seen comparing ABX-treated and non-ABX exposed groups. In mice that received cF8, a drop in mean FVIII:C occurred due to inhibitor formation, with total loss of expression in 50% ABX-treated and 28% untreated mice.

Conclusion(s): Liver-directed transgene expression was not influenced by gut microbiome manipulation. Increased incidence of FVIII inhibitor was seen in mice with dysbiosis.