A Reverse Transcription Nucleic-Acid-Based Barcoding System for In Vivo Measurement of Lipid Nanoparticle mRNA Delivery

Abstract

Lipid nanoparticles (LNPs) are the most extensively validated clinical delivery vehicles for mRNA therapeutics, exemplified by their widespread use in the mRNA COVID-19 vaccines. The pace of lipid nanoparticle (LNP) development for mRNA therapeutics is restricted by the limitations of existing methods for large-scale LNP screening. To address this challenge, we developed Quantitative Analysis of Reverse Transcribed Barcodes (QuART), a novel nucleic-acid-based system for measuring LNP functional delivery in vivo. QuART uses a bacterial retron reverse transcription system to couple functional mRNA delivery into the cytoplasm with a cDNA barcode readout. Our results demonstrate that QuART can be used to identify functional mRNA delivery both in vitro in cell culture and in vivo in mice. Multiplexing of QuART could enable high-throughput screening of LNP formulations, facilitating the rapid discovery of promising LNP candidates for mRNA therapeutics.

Figure 1

Overview of the reverse transcription barcoding system. Delivered mRNA will express reverse transcriptase inside of the cytoplasm. RNA barcodes will be reverse transcribed into cDNA barcodes by the reverse transcriptase. mRNA that does not enter the cell remains unchanged and is removed through an RNase digest. The cDNA barcodes are then sequenced to identify the mRNA that entered the cytoplasm.

Figure 2

A) Schematic of the reverse transcription barcoding mRNA. The RNA barcode template is included in the reverse transcriptase mRNA in the 3′ UTR. B) 10% TBE polyacrylamide gel image of the test results from the retrovirus reverse transcription mRNA transfection. The barcodes are visible as a 104bp band. The positive control is the PCR product using the barcode template oligonucleotide as a template. A no-template PCR control was included to check for primer dimers or contamination. C) Diagram summarizing the experimental procedures to test the effect of the functional delivery of the components. The reverse transcriptase and retron reverse transcription template RNA barcode are added as naked RNA or in complex with transfection reagent. D) 2% TBE agarose gel image of the test results from cDNA barcode expression mechanism test. The barcode is present as a 104bp band only when both RT mRNA and barcode template are transfected together. A no-template PCR control is included to check for primer dimers or contamination.

Figure 3

A) Overview of the cDNA quantitation experiments by qPCR. B) Bar graph illustrating the normalized quantities of cDNA barcodes relative to mRNA dosage. The amount of cDNA barcodes expressed at the 24-h time point is significantly higher (p = 0.03, Welch’s t-Test) than the amount of cDNA barcodes at the 48-h time point. C) Scatter plot showing the correlation between cDNA barcode concentration and mRNA dosage. The cDNA barcode quantity shows a strong positive correlation with mRNA dosage (R² = 0.81).

Figure 4

A) Overview of the barcode multiplexing experiment. B) Comparison of the expected barcode counts and actual barcode counts. The actual distribution of the barcodes does not differ significantly from the expected distribution of barcodes (p = 1.0, Welch’s t-Test n = 2) (Kullerbeck–Leibler divergence = 0.05).

Figure 5

A) Overview of modifications designed to enhance cDNA expression in vivo. B) Overview of the experimental procedure to test the detection limit of the reverse transcription mRNA variants in vivo. C) 10% TBE polyacrylamide gel of the in vivo cDNA barcode expression results. Non/Uninjected mouse (UT) shows no cDNA expression while all doses of mRNA tested showed cDNA expression.