Self-Amplifying mRNA Enhances Transamniotic Fetal mRNA Delivery

Intraamniotic injection of encapsulated linear mRNA routes to the fetus after placental uptake but has a limited half-life and translation. Self-amplifying mRNA (saRNA) is a synthetic mRNA containing a replicase sequence and gene of interest (GOI) with replicase-targeting motifs that allow for the amplification of both the full saRNA and GOI. We compared the fetal distribution of saRNA and linear mRNA with the same GOI after intraamniotic injections in a rat model. Fetuses (n = 99) from seven Sprague-Dawley dams were divided into three groups based on volume-matched intraamniotic injections performed on gestational day 17 (E17; term = E21-E22). Two groups received firefly luciferase mRNA either in linear or self-amplifying form, both encapsulated by lipopolyplex. The third group received lipopolyplex without mRNA (controls). All groups were euthanized at term, while only saRNA and controls were euthanized on postnatal day 7 or 14. Luciferase activity was screened via microplate luminometry at multiple fetal and neonatal anatomical sites. Significantly higher luminescence compared to controls was detected at term at 9 anatomical sites (placenta, umbilical cord, gestational membranes, amniotic fluid, liver, heart, lung, kidney, and thymus; p = 0.039-< 0.001) in the saRNA group but at only 1 site (umbilical cord; p < 0.001) in the linear mRNA group. No significant differences with the controls were detected at any other timepoint. Self-amplifying mRNA leads to extended and more robust fetal and fetal annex translation of its encoded protein when compared with linear mRNA after transamniotic delivery. Self-amplifying mRNA may maximize the therapeutic potential of transamniotic fetal mRNA therapies.