Ionizable lipid nanoparticles encapsulating barcoded mRNA for accelerated in vivo delivery screening

Purpose: LNP libraries are usually screened in vitro, which poorly predicts in vivo mRNA delivery. A high-throughput in vivo platform was needed to screen many LNP formulations simultaneously and identify lead carriers for functional mRNA delivery.

Hypothesis: If functional mRNA is engineered with a barcode and UMI in its 3′ UTR, then multiple barcoded mRNA-LNP formulations can be pooled, injected intravenously, and quantified simultaneously by deep sequencing. This b-mRNA platform should better predict functional mRNA delivery than DNA-barcoded LNPs because the cargo more closely mimics therapeutic mRNA size and structure.

Aims: Synthesize functional barcoded mRNA (b-mRNA) encoding luciferase with barcode and UMI sequences. - Formulate and characterize a mini-library of b-mRNA LNPs. - Screen multiple b-mRNA LNP formulations simultaneously in mice for delivery to liver, spleen, and other organs. - Validate deep-sequencing delivery results against functional luciferase and EPO expression. - Compare b-mRNA LNPs with b-DNA LNPs and assess which better predicts functional mRNA delivery.

Delivery system: Platform: Ionizable lipid nanoparticles (LNPs) encapsulating barcoded mRNA. - Ionizable lipid: C12-200. - Excipients: DOPE, cholesterol, C14-PEG2000. - Payload: In vitro transcribed b-mRNA encoding firefly luciferase, with barcode, UMI, T7 promoter, PCR handle, and poly(A) tail; m5C-modified b-mRNA used for most studies. - Formulation: Microfluidic mixing of aqueous mRNA phase and ethanolic lipid phase. - Targeting ligand: None. - Route: Intravenous tail-vein injection in C57BL/6 mice. - Key lead formulation: F13; also compared F01, F04, F06, F09, F16.

Approach: In vitro: bEnd.3 cells for b-mRNA transfection; m5C vs Ψ modification comparison. - In vivo: C57BL/6 mice; pooled b-mRNA LNPs injected IV at 0.25 µg per formulation for screening; tissues harvested 4 h post-injection. - Dose–response: 17–1000 ng b-mRNA per formulation to test detection range. - Validation: F01 and F13 formulated with luciferase or EPO mRNA and injected separately; luminescence and serum EPO measured. - b-DNA comparison: Same 16 LNP formulations encapsulating b-DNA and screened identically.

Key methods: DLS for hydrodynamic diameter and PDI. - Cryo-TEM for LNP morphology. - RiboGreen assay for mRNA encapsulation efficiency. - Deep sequencing of barcodes for b-mRNA and b-DNA quantification in tissues. - IVIS imaging for in vivo luciferase expression. - ELISA for serum EPO. - Linear regression for dose–response and b-mRNA vs b-DNA correlations.

Key results: b-mRNA could be detected in liver at doses as low as 17 ng; b-mRNA delivery was dose-dependent with linear correlation R² = 0.9646. - 16 b-mRNA LNP formulations were pooled and screened simultaneously; F13 showed high delivery to liver and spleen, and F14–F16 showed high delivery to most tissues. - F13 produced higher liver and spleen luciferase expression than F01; F13 also produced significantly higher serum EPO than F01. - b-DNA LNPs showed weak correlation with b-mRNA LNPs for liver (R² = 0.0164) and spleen (R² = 0.2505) delivery. - F04 was a lead formulation for b-DNA delivery but not for b-mRNA; F13 was more potent than F04 for EPO production, indicating b-mRNA better predicts functional mRNA delivery. - LNP sizes were ~74–91 nm; most had PDI 0.174–0.233; encapsulation efficiency >85% for 11 of 16 formulations.

Interpretation: b-mRNA LNPs enable simultaneous in vivo screening of many mRNA formulations and can identify lead LNPs for functional mRNA delivery. Because b-mRNA mimics therapeutic mRNA size and structure better than b-DNA, it is a more predictive “first-pass” screen for mRNA-LNP development.

Limitations: Only mice; no large-animal validation. - Single 4 h timepoint for most delivery measurements. - No disease model or therapeutic efficacy endpoint beyond reporter/EPO expression. - No long-term safety, toxicity, or repeated-dose evaluation. - b-mRNA is a proxy and may not fully replicate every therapeutic mRNA. - Screening used one ionizable lipid class (C12-200) and a small 16-formulation mini-library. - Delivery across different organs cannot be directly compared with the quantification method used.