OC 12.1 - Transgene expression patterns after AAV8 delivery using hepatocyte promoter elements reveal expression in both hepatocytes and liver sinusoidal endothelial cells (LSECs)

Sunday, July 10, 2022

10:45 AM – 11:00 AM

Location: ExCel Center, S11A

Presentation Format: In-Person

Presenting Author(s)

JL

Jonathan R. Lindgren, B.S.

Research Tech II
Children's Hospital of Philadelphia Center for Cellular and Molecular Therapeutics
Philadelphia, Pennsylvania, United States

Co-Author(s)

DS

Denise E. Sabatino

Research Associate Professor of Pediatrics
Children's Hospital of Philadelphia Center for Cellular and Molecular Therapeutics
Philadelphia, Pennsylvania, United States
GN

Giang Nguyen

Research Associate
Children's Hospital of Philadelphia Center for Cellular and Molecular Therapeutics
Philadelphia, Pennsylvania, United States
Antonia Follenzi, MD, PhD

Full professor
Department of Health Sciences, University of Piemonte Orientale, Novara, Italy
Novara, Piemonte, Italy
AH

Allysen Henriksen

Research Tech II
Children's Hospital of Philadelphia Center for Cellular and Molecular Therapeutics
Philadelphia, Pennsylvania, United States
RF

Rosella Fama

Research Associate
Children's Hospital of Philadelphia Center for Cellular and Molecular Therapeutics
Philadelphia, Pennsylvania, United States

Background: Adeno-associated virus (AAV)-mediated gene therapy for hemophilia A (HA) is actively in clinical development. Due to the packaging constraints of the AAV vector, minimized promoter elements derived from hepatocyte specific genes have been developed to direct factor VIII (FVIII) expression to the liver. The human alpha-one antitrypsin/ApoE promoter-enhancer (hAAT/ApoE) (Pasi 2020) and the transthyretin (TTR) promoter (George 2021) are used in HA clinical studies to achieve liver-specific FVIII expression. While the primary endogenous site of FVIII synthesis is liver sinusoidal endothelial cells (LSECs), the cellular profile of transgene expression in the liver after AAV gene therapy utilizing these promoters is not known.

Aims: Determine the cellular specificity of the hAAT/ApoE and TTR promoters after systemic delivery of AAV8 vectors.

Methods: AAV8-TTR-GFP (n=4) and AAV8-hAAT/ApoE-GFP (n=3) were administered intravenously to HA-CD4KO mice (1e11 vg/mouse). LSECs and hepatocytes were analyzed for GFP expression by flow cytometry (FACS). CD31 and CD146 were used to isolate and label LSECs for flow analysis, while albumin was used to label hepatocytes. Immunofluorescence (IF) staining was conducted in parallel to evaluate GFP expression in LSECs and hepatocytes.

Results: AAV8-TTR-GFP produced GFP expression in a similar proportion of hepatocytes (83.3% GFP+/albumin+ +/- 12.8%) and LSECs (71.8% GFP+/CD146+/CD31+ +/- 15.9%)(Figure 1). Delivery of AAV8-hAAT/ApoE-GFP also resulted in GFP expression in both hepatocytes (76.2% GFP+/albumin+ +/- 15.4%) and LSECs (87.9% GFP+/CD146+/CD31+ +/-6.4%). Immunofluorescent staining of liver confirms FACS data, showing transgene expression in both hepatocytes and LSECs.

Conclusion(s): AAV8 constructs under control of minimized hAAT/ApoE and TTR promoter elements drive transgene expression in hepatocytes and LSECs. Further studies are required to understand the biological consequences of expressing FVIII in hepatocytes and LSECs after AAV gene therapy.