OC 12.5 - Liver Sinusoidal Endothelial Cells Targeted with Ultrasound Mediated Gene Delivery Shows Long Term FVIII Expression in Hemophilia A Mice

Sunday, July 10, 2022

11:45 AM – 12:00 PM

Location: ExCel Center, S11A

Presentation Format: In-Person

Presenting Author(s)

SL

Savannah M. Lawton, BS

Research Scientist
Seattle Children's Research Institute
Seattle, Washington, United States

Co-Author(s)

MM

Megan Manson

Research Scientist I
Seattle Children's Research Institute - Seattle, Washington
Seattle, Washington, United States
MF

Meng-Ni Fan

Research Scientist II
Seattle Children's Research Institute
Seattle, Washington, United States
TC

Ting-Yen Chao

Research Scientist
Seattle Children's Research Institute
seattle, Washington, United States
PK

Peter Kim

Research Scientist II
Seattle Children's Research Institute
Seattle, Washington, United States
CC

Carley Campbell

Research Scientist II
Seattle Children's Research Institute
Seattle, Washington, United States
CC

Chun-Yu Chen

Research Scientist II
Seattle Children's Research Institute
seattle, Washington, United States
XC

Xiaoche Cai

Research Scientist I
Seattle Children's Research Institute
seattle, Washington, United States
AV

Amber Vander Kooi

Research Scientist II
Seattle Children's Research Institute
Seattle, Washington, United States
CM

Carol Miao

Principal Investigator
Seattle Children's Research Institute
Seattle, Washington, United States

Background: Ultrasound-mediated gene delivery (UMGD) with microbubbles (MBs) is a potentially effective method of non-viral gene delivery to treat hemophilia A (HA). Previously, we found liver sinusoidal endothelial cells (LSECs) could be targeted with UMGD at a lower power than hepatocytes. We hypothesized that gene expression with lower energy conditions would be comparable to higher energy conditions, however, with less damage to the liver or induction of anti-FVIII inhibitors.

Aims: In this study, we explored US conditions that target LSECs with UMGD to induce sustained FVIII expression in HA mice.

Methods: Mouse liver was injected via the portal vein with a combination of plasmid DNA and MBs. Simultaneously, a pulsed therapeutic US transducer was applied to the liver surface for one minute at 1.1 MHz frequency and 14 Hz PRF. HA mice were separated into two different US condition groups, low energy (LE; 50 W/cm2, 150us PD) targeting predominantly endothelial cells, or high energy (HE; 110 W/cm2, 150 us PD) targeting predominantly hepatocytes. A high-expressing, endothelial-specific hFVIII plasmid, pUCOE-ICAM2-hF8/N6-X10, was used. To study hFVIII activity over 84 days, APTT was run on plasma. Livers were sectioned, stained, and imaged with a Leica DM6000 fluorescent microscope.

Results: FVIII activity levels for both the LE and HE groups stabilized around 10% at 84 days post-treatment (Figure 1). RNAscope©® Multiplex Fluorescent staining showed colocalization of hFVIII and Lyve-1 (LSEC marker) at D7 and D120. Transaminase levels indicated the LE group had lower transient liver damage than the HE group in the first week with both groups returning to baseline by week two.

Conclusion(s): We show LSECs can be targeted for transfection using lower energy than a previously established hepatocyte targeting condition and produced FVIII activity over 84 days. The ability to target different cell types with UMGD can be applied to a multitude of genetic conditions.