In Vivo Methodology Measures Nanoparticle Supply Effectivity

Measuring the efficiency with which lipid nanoparticles release genetic material in gene therapy provides a roadmap for more efficient drug delivery.

Many gene therapies rely on microscopic spherical lipid nanoparticles (LNPs), which encase and shield the therapeutic genetic material from degradation.¹ Upon delivery, cells internalize these carriers and trap them into endosomes, which mature and fuse with lysosomes—forming structures called endolysosomes—to degrade the particles.

While tailored LNPs release their payload into the cytoplasm before they get degraded, the efficiency of this process—called endosomal escape—remains as low as two percent.² This highlights the need for more effective delivery vehicles, but a lack of reliable tools to measure endosomal escape in vivo has impeded these advancements.

Now, scientists have developed a method to quantify the endosomal escape of LNP-delivered genetic material in mice without using artificial reporters or microscopy.³ Their approach to measure LNP-encapsulated DNA barcodes, published in Nature Biotechnology, provides a framework to investigate intracellular trafficking and biodistribution of gene therapy tools under physiological conditions.

“Once you can measure something, you can design around it,” said study coauthor Gaurav Sahay, a drug delivery scientist at Oregon State University, in a statement. “Designs based on our measurements allow for new lipid nanoparticles capable of much more efficient delivery.”

Sahay and his team started out by designing and synthesizing LNPs carrying mRNA encoding a bioluminescent reporter. They shortlisted the most effective formulations by tracing the highest fluorescence that localized to the target organ—the liver—after injecting them into mice.

The researchers then investigated whether their LNPs could successfully deliver CRISPR-Cas9 gene-editing tools. They injected mice with machinery to disrupt the gene encoding transthyretin (TTR), a transport protein, and carried out next-generation sequencing, which revealed successful editing of nearly 20 percent of the DNA. Diminished TTR levels in the serum further confirmed successful gene editing.

Encouraged by this, Sahay and his team decided to quantify endosomal escape in vivo using these LNPs. They used mice engineered to carry endolysosome-specific tags, which enabled them to purify these organelles. The researchers injected these animals with LNPs encapsulating unique DNA barcodes, which they then measured using PCR.

The researchers observed that the amount of DNA barcodes within endolysosomes declined within two hours of injection, suggesting that endolysosomal escape occurs within that window. They then compared the quantity of barcodes localized within the endolysosomes and whole liver tissue within that timeframe.

“That allowed us to quantify how efficiently different nanoparticle designs release their cargo,” said study coauthor Antony Jozić, a graduate student in Sahay’s lab, in the statement. Their calculations revealed that the endolysosomal escape of their LNPs was eight percent at 30 minutes, which dropped to six percent one hour post administration.

“This insight resolves a longstanding challenge in our field, to track genetic material inside the subcellular compartments within the cell in a living organism and provides a road map for improving RNA and gene-editing medicines and reducing off-target effects,” said Sahay.