Benchmarking RNA Delivery Performance: Toward Standardized Metrics For Translational Success

By Jyotsna Jajula, research associate, Wayne State University

The rapid rise of RNA-based therapeutics, including messenger RNA (mRNA) vaccines, small interfering RNA (siRNA), and circular RNA (circRNA), has brought renewed attention to a long-standing challenge: delivering RNA precisely, efficiently, and safely to target tissues. Despite significant advances in molecular engineering and formulation chemistry, the delivery vehicle remains a critical bottleneck to translational success. Lipid nanoparticles (LNPs), polymers, and virus-like particles (VLPs) each offer unique advantages, but the absence of standardized performance benchmarks continues to hinder meaningful comparison, optimization, and regulatory alignment.^1,2

Currently, delivery efficiency is assessed using fragmented and often inconsistent metrics,² ranging from cellular uptake and internalization rates to reporter gene expression or protein translation. These outcomes are highly context-dependent and frequently conflate payload potency with delivery success. Without decoupling these variables, it becomes difficult to pinpoint failure modes or to compare novel delivery systems against well-characterized reference platforms.^1,3

To address this issue, we propose a harmonized delivery benchmarking framework centered on four quantifiable and orthogonal performance domains:

1. cellular uptake,
2. endosomal escape,
3. cytoplasmic RNA stability, and
4. tissue-specific biodistribution.

By establishing shared protocols and performance thresholds across these domains, researchers can better interpret preclinical outcomes, guide carrier optimization, and accelerate the development of clinic-ready RNA delivery systems.^1,4

Why Benchmarking RNA Delivery Matters

While RNA therapeutics are advancing rapidly, the lack of standardized delivery benchmarks remains a significant blind spot in preclinical development. Laboratories often rely on different cell types, animal models, reporter systems, and time points to evaluate delivery performance. As a result, comparing one platform’s efficacy against another becomes inherently flawed. For instance, an LNP formulation tested in human hepatocytes may appear more effective than a polymeric carrier evaluated in murine fibroblasts, but such comparisons are biologically invalid without proper normalization.^2,3

Even within the same modality, delivery performance can vary based on formulation parameters, batch-to-batch variability, and the nature of the RNA cargo (e.g., mRNA, siRNA, circRNA). Many published studies report qualitative or single-endpoint outcomes, such as protein expression, which conflate delivery efficiency with downstream translation or RNA stability.^5,6 Without isolating these steps, it becomes impossible to determine whether a delivery system failed due to insufficient uptake, endosomal trapping, or cytoplasmic degradation.

These inconsistencies hinder not only platform optimization but also complicate regulatory submissions. The FDA and EMA expect more rigorous, quantitative characterization of delivery mechanisms,⁷ particularly for next-generation RNA platforms targeting extrahepatic or immunologically privileged tissues. In this evolving landscape, developers who adopt validated, comparative benchmarks will be better positioned to demonstrate functional superiority and advance more confidently through regulatory review.

A benchmarking framework that disaggregates delivery stages and relies on cross-platform assays will empower developers to identify performance bottlenecks, guide rational design, and set credible performance thresholds. Such a shift, from exploratory trial-and-error to mechanistic, data-driven development, is essential for RNA therapeutics to expand beyond hepatic delivery and into emerging areas such as oncology, neurology, and immunotherapy.⁹

Four Pillars of RNA Delivery Performance

Cellular Uptake

Efficient cellular uptake is the critical first step in any RNA delivery pathway. However, many studies continue to rely on indirect metrics such as downstream protein expression, which can obscure whether the RNA was ever successfully internalized. Quantifying uptake independently of expression is essential to distinguish delivery performance from functional RNA output.⁴

Flow cytometry and confocal microscopy using fluorescently labeled RNA or nanocarriers remain common techniques.⁷ Recently, more advanced methods, including click chemistry, based probes, and pH-sensitive dyes, have emerged to distinguish surface binding from true internalization.¹⁰ These tools can also help identify uptake pathways, such as clathrin-mediated endocytosis, caveolin-dependent uptake, or macropinocytosis.¹¹ To enable standardized comparisons, we propose benchmarking uptake using percent internalization in a reference cell line (e.g., HepG2 or HeLa) at a defined post-delivery time point (e.g., 4 hours), quantified using validated fluorescent conjugates.

Endosomal Escape

Once internalized, RNA therapeutics must escape the endosomal compartment to reach the cytoplasm, a key bottleneck for effective delivery. Without escape, most cargo is degraded in lysosomes.⁵

New assays now allow direct visualization of escape events. For example, galectin-8 recruitment marks disrupted endosomes,¹² while split-GFP or split-luciferase systems activate only upon cytoplasmic exposure.¹³ The field is also exploring quantitative indices such as the Endosomal Escape Index (EEI) to compare platform efficiency across conditions.¹⁴ A robust benchmarking strategy should use standardized assays across consistent cell types and normalize results to RNA dose and exposure time.¹⁵

Cytoplasmic RNA Stability

Successful endosomal escape does not guarantee RNA functionality.^2,5 Once in the cytoplasm, RNA must remain stable long enough to be translated or engage the RNAi machinery. However, unmodified RNA is highly vulnerable to nucleases and innate immune sensors.²

Stability can be assessed using:

• RT-qPCR for delivered sequence degradation,¹⁶
• Live-cell imaging of fluorescently tagged RNA,¹⁸
• Nanopore sequencing to distinguish intact vs. fragmented RNA molecules.¹⁹

Measuring RNA half-life in the cytoplasm and normalizing it to controls (e.g., pseudouridine-modified vs unmodified RNA) can provide insight into carrier protection capabilities. Additionally, RNA granules and P-body sequestration, increasingly implicated in non-productive delivery, represent an underexplored but important factor in functional delivery loss.¹⁷

Tissue-Specific Biodistribution

Tissue specificity remains one of the most pressing delivery challenges. While LNPs have demonstrated remarkable success in hepatocyte targeting, extrahepatic delivery to lungs, tumors, muscle, or the brain remains unpredictable and poorly benchmarked.^21,22

Modern tools are enabling finer resolution of RNA biodistribution profiles. These include:

• In vivo imaging systems (IVIS), ²³
• Radiolabel tracking,²⁴ and
• Sequencing-based barcoding (e.g., FIND-seq, PEGylated LNP libraries)²⁰

These methods quantify localization across organs, time points, and dose levels.

A widely accepted benchmark, such as percent injected dose per gram of tissue (%ID/g), could serve as a normalized metric, provided dosing routes, cargo types, and  standardized timelines.²⁵

Toward A Harmonized Evaluation Framework

Despite decades of formulation innovation, the RNA delivery field still lacks a unifying language for measuring success. A harmonized benchmarking framework, rooted in reproducible, quantitative assays, would allow researchers and companies to compare platforms with clarity and confidence.²⁶

What would such a framework require? First, standard reference cell lines and animal models would require agreement across academic, biotech, and regulatory stakeholders. Just as the RECIST criteria transformed oncology trial interpretation, RNA delivery needs reference thresholds. For example, a benchmark LNP showing ≥60% uptake in HepG2 cells or ≥5%ID/g in liver 24 hours post-injection. These values can serve as internal controls or gates in screening pipelines.

Second, reporting guidelines must evolve. Journals and funding bodies could require that studies report delivery efficacy across multiple performance domains, uptake, escape, stability, biodistribution, rather than single outcome metrics. This shift would force greater assay transparency and push the field toward standardization.^26,27

Finally, public-private consortia could play a leading role in organizing delivery benchmark datasets, similar to the way the NIH Common Fund has supported data standards in the Human BioMolecular Atlas Program (HuBMAP) or SEQC for RNA-seq. Initiatives that open-source comparative data on delivery platforms would accelerate progress across academia and industry.²⁸

A harmonized framework would not only improve reproducibility, but also change the way we select, refine, and approve delivery technologies. In a competitive therapeutic landscape, clarity around delivery performance could become the deciding factor between a shelved candidate and a successful one.²⁹

Conclusion: Enabling Translational Confidence

As RNA therapeutics push into new disease areas and delivery challenges become more complex, standardized benchmarking is no longer optional; it is essential.³⁰ Without shared metrics, we risk misinterpreting efficacy, duplicating failed efforts, and slowing innovation.

By centering evaluation around four quantifiable delivery domains – cellular uptake, endosomal escape, cytoplasmic RNA stability, and tissue-specific biodistribution – researchers can identify true strengths and weaknesses in their platforms. Just as pharmacokinetics and toxicity thresholds are core to small molecule development, delivery performance must become a core parameter for RNA formulations.³¹

A harmonized framework will not only improve preclinical rigor, it will also build translational confidence for regulators, clinicians, and patients.³² The field is ready for benchmarking. The question is: will we take the leap toward comparability, or continue to navigate blindfolded?

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About The Expert

Jyotsna Jajula is a Research Associate II in the Biosciences Division at SRI International. Her work broadly explores RNA delivery mechanisms in oncology cell models, with a focus on internalization and cytoplasmic fate of therapeutic peptides. She holds a master’s degree in pharmaceutical sciences and has prior research experience in lipid nanoparticles, RNA stability, and biodistribution strategies across oncology, immunology, and gene-therapy applications.