Molecular therapy : the journal of the American Society of Gene TherapyApr 15, 2026

How Messenger and Guide RNA Amounts in Fatty Nanoparticles Affect Gene Editing Efficiency in Living Organisms

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗

Updated Apr 16, 2026

Abstract

Co-packaging of mRNA and guide RNA in lipid nanoparticles results in distinct subpopulations with varying RNA distributions.

Four distinct subpopulations of lipid nanoparticles were identified: co-encapsulated (50.7-60.4%), gRNA-only (30.0-36.5%), mRNA-only (2.0-3.4%), and empty (4.2-13.8%).
There is significant variability in RNA copy number within each subpopulation of lipid nanoparticles.
Lipid nanoparticle formulation and mixing methods impact payload distribution, showing a negative correlation between empty particles and RNA loading.
In mice, lipid nanoparticles with higher mRNA and gRNA loads exhibited 1.5-fold greater gene-editing activity, achieving 55.4% indels compared to 36.3% with lower loads.
The findings suggest that payload distribution could influence the effectiveness of gene-editing therapies.

Simplified

Lipid nanoparticles (LNPs) are among the most advanced non-viral vectors for CRISPR-based gene-editing therapeutics. Co-packaging of messenger RNA (mRNA) and guide RNA (gRNA) inherently produces heterogeneous payload distributions. The impact of this heterogeneity on editing performance remains unclear. Here, we utilize cylindrical illumination confocal spectroscopy (CICS) for single-particle interrogation of ALC-0315 and DLin-MC3-DMA LNPs prepared by three different mixing methods. CICS resolves four distinct subpopulations: co-encapsulated (50.7-60.4%), gRNA-only (30.0-36.5%), mRNA-only (2.0-3.4%), and empty (4.2-13.8%), and uncovers broad, particle-to-particle variability in RNA copy number within each class. Structure-function analysis reveals that LNP formulation and mixing processes influence payload distribution, resulting in a negative correlation between the fraction of empty LNPs and RNA loading per particle. We further investigated the correlation between these quality attributes and therapeutic performance. In mice, ALC-0315 LNPs carrying higher cargo loads (9.8 vs. 8.0 mRNA copies and 25.4 vs. 20.3 gRNA copies per co-encapsulated particle) yielded 1.5-fold higher in vivo editing activity (55.4% vs. 36.3% indels) despite nearly identical biophysical characteristics including LNP size and RNA encapsulation. These results establish payload distribution as a potential determinant of gene-editing potency and demonstrate single-particle CICS as a powerful tool for rational design of multi-component nucleic acid delivery systems.

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Competing interests

Declaration of interests S.L., H.-Q.M., and T.-H.W. are inventors of a patent application covering the CICS technique described in this paper, filed through and managed by the Johns Hopkins Office of Technology Ventures. L.B.T., C.D., C.C., S.H., M.K., and S.Z. declare an association with Arbor Biotechnologies. S.L., H.-Q.M., and T.-H.W. are co-founders of CICS Analytics Corp., which is commercializing the CICS technology described here.

PubMed

How Messenger and Guide RNA Amounts in Fatty Nanoparticles Affect Gene Editing Efficiency in Living Organisms

Abstract

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