Amniotic fluid stabilized lipid nanoparticles for in utero intra-amniotic mRNA delivery

Purpose: Congenital protein deficiencies often begin irreversible pathology in utero, but postnatal treatments are limited. In utero mRNA delivery could enable protein replacement before disease onset, yet LNP stability in fetal amniotic fluid and how it affects mRNA delivery had not been previously investigated.

Hypothesis: Evaluating LNP stability in amniotic fluid can identify novel LNPs well-suited for prenatal gene therapies; LNPs that are stable in amniotic fluid will enable potent intra-amniotic mRNA delivery in utero.

Aims: Design a library of 16 ionizable LNPs using orthogonal design of experiments (DOE) with varying molar ratios of four excipients. - Assess ex vivo LNP stability in mouse serum and mouse, sheep, pig, and human amniotic fluids using dynamic light scattering (DLS). - Evaluate in vitro luciferase mRNA delivery and cytotoxicity in HeLa cells and primary fetal lung cells. - Test a stable and an unstable LNP for in utero intra-amniotic luciferase mRNA delivery in mice.

Delivery system: Platform: Ionizable lipid nanoparticles (LNPs) for mRNA delivery. - Key material: Ionizable lipid B-4 (synthesized via Michael addition) or C12-200; excipients: DOPE, cholesterol, C14-PEG2000. - Payload: Nucleoside-modified (m1Ψ) firefly luciferase mRNA with 101-nt poly(A) tail. - Formulation: 10:1 weight ratio of ionizable lipid to mRNA; library varied molar ratios of B-4, DOPE, cholesterol, and PEG. - Targeting: No active targeting ligand; intra-amniotic injection for fetal delivery. - Physicochemical properties: Sizes 46–153 nm; PDI 0.18–0.61; encapsulation 16–96%; pKa 6.03–6.63; zeta potential −7.4 to +25 mV.

Approach: In vitro: HeLa cells and primary fetal lung cells from E16 Balb/c mice; LNP treatment at 10–100 ng mRNA per 20,000 cells (primary) or 50 ng per 10,000 cells (HeLa). - In vivo: Time-dated pregnant Balb/c mice at gestational day E16; intra-amniotic injection of 30 µL LNPs (325 ng/µL) or PBS; n = 5 fetuses per group (A12, A4, PBS). - Fluid stability: 16 LNPs incubated in 50% (v/v) mouse serum or mouse, sheep, pig, or human amniotic fluid for 30 min at 37°C. - Disease context: Proof-of-concept for prenatal gene therapy of congenital disorders.

Key methods: DLS for size and PDI; zeta potential measurements. - RiboGreen assay for mRNA encapsulation efficiency; pKa by TNS fluorescence. - TEM for morphology in PBS and mouse amniotic fluid. - Sepharose CL-6B chromatography with protein quantification for protein corona analysis. - Luciferase luminescence assay for in vitro delivery; CellTiter-Glo for viability. - IVIS imaging for in utero fetal and organ bioluminescence.

Key results: LNP A12 was most stable in mouse amniotic fluid; A1 was least stable. A12 had significantly higher bound protein content than A1 (p < 0.0021). - Percent change in PDI inversely correlated with in vitro luciferase expression; A12 had the highest in vitro delivery among stable LNPs. - In primary fetal lung cells, A12 produced significantly higher luciferase expression than A4 at all doses (10, 25, 50, 100 ng; p < 0.05). - In utero, A12 yielded significantly higher fetal bioluminescence than A4 and PBS (p < 0.05); A4 was not significantly different from PBS. - A12 showed luminescence in fetal lung, intestine, and liver; A4 and PBS showed no organ luminescence.

Interpretation: The authors conclude that ex utero LNP stability in amniotic fluid can predict in utero intra-amniotic mRNA delivery. Stable LNPs such as A12 are promising for prenatal gene therapy, and this screening approach may accelerate development of LNPs for congenital disease treatment.

Limitations: In vivo delivery was tested only in mice; no large-animal or human in utero delivery validation. - Human amniotic fluid was used only for stability screening, not for delivery. - No disease model; only luciferase reporter mRNA. - Variability in fetal luciferase expression was noted. - Short-term expression (4 h post-injection) and no long-term safety/efficacy data. - Encapsulation efficiency ≤75% for several LNPs limited comparison.