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.

Figure 1

HA mice in both the LE and HE ultrasound treatment groups had hFVIII activity levels between 5-25% with stabilization of around 10% at day 84.

Figure 2

Livers from LE mice harvested at day 7 -A- and day 120 -B- showed expression of hFVIII mRNA shown in green, and Lyve-1 mRNA, an endothelial-specific marker, in red. Colocalization appears yellow.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/liver-sinusoidal-endothelial-cells-targeted-with-ultrasound-mediated-gene-delivery-shows-long-term-fviii-expression-in-hemophilia-a-mice/