PROTEIN BARCODING - Streamlining mRNA Therapeutic & Vaccine Development

INTRODUCTION

Nucleic acid-based vaccines and therapeutics, including mRNA-based platforms, represent a groundbreaking advance in healthcare. A critical determinant of therapeutic efficacy of these treatments is the delivery system, which ensures precise targeting to the intended tissues while also minimizing degradation and off-target effects.

Lipid nanoparticles (LNPs) have emerged as a powerful tool for delivery due to their low immunogenicity compared to viral vectors, along with their ability to encapsulate and protect nucleic acids and facilitate their entry into cells. LNPs are used in the COVID-19 mRNA vaccines, for siRNA delivery, and in numerous clinical trials with a range of payloads. However, the majority of these applications are in liver-focused disease areas simply be­cause that is where LNPs accumulate in the body when adminis­tered systemically.¹

 TARGETING LNP DELIVERY

Even subtle changes in LNP lipid composition, ratios, particle size, or surface modifications can significantly alter how the LNP behaves in vivo and thus affect biodistribution, immunogenicity, and expression kinetics. As mRNA therapies move beyond the liver and into applications targeting other tissues, customized LNP formulations are essential for precise and effective delivery.

Directing mRNA to different tissues thus requires high-throughput screening of a vast number of mRNA-LNP formula­tions. In general, the LNP screening process involves formulating a library of different LNP compositions and administering them, often one at a time, in animal models. Tissues are collected and mRNA expression is measured, typically through protein output or reporter assays (Figure 1). This approach is labor-intensive, low-throughput, and expensive, as each formulation must be evaluated in separate animals or animal groups.

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The specific steps in the workflow for creating and screening mRNA-LNPs vary depending on the specific assay require­ments, but generally include the following:

Select, synthesize, and characterize the mRNA payload: Confirm the mRNA se­quence after synthesis, determine purity, and characterize/optimize the 5’ cap and 3’ poly(A) tail.

Select and formulate lipid components: To achieve maximum efficacy, each LNP formulation must be optimized for specific biophysical characteristics such as shape, size, payload density, and surface charge.

Select the screening method: Select in vitro (2D or 3D cell culture) or in vivo (an­imal models) methods.

Select the screening readout: Fluores­cence or next-generation sequencing, de­pending on the reporter used in the mRNA construct.

Encapsulate the mRNA into the LNP: The purified mRNA is combined with the lipid nanoparticle solution, allowing the mRNA to be encapsulated within the LNP’s hy­drophobic core.

Quality control and characterize the final product: Perform analytical tests to assess the size, stability, encapsulation ef­ficiency, and purity of the mRNA-LNP for­mulation.

Perform the screen: Based on the method and readout selected.

Draw conclusions: Identify the top candi­dates from the screen and feed the results back into mRNA and lipid formulations for further studies and optimization.

While different tools are available to support nanoparticle design, formulation, and quality control (steps 1-6), the screen­ing step (step 7), still faces limitations with current methods, which include next-gen­eration sequencing (NGS), mass spec­trometry, flow cytometry, ELISA, and Western blotting.^2-7  For example, using NGS for screening is efficient because it allows for the analysis of multiple mRNAs in a single animal. However, a significant limitation of this approach is that it meas­ures delivery – ie, the presence of mRNA – rather than translation of the mRNA into the desired protein.5,6

This can lead to inaccurate results and erroneous candidate selection.

In contrast, screening that uses a pro­tein-level readout, such as mass spectrom­etry (MS) or flow cytometry, provides evidence that the mRNA was both deliv­ered and translated effectively.^7,8

Limited tools are available for multi­plexing at the protein level, which in­creases costs and reduces efficiency; in the case of flow cytometry, this also results in low sensitivity. Therefore, new tools that fa­cilitate multiplex pooled screening with a direct protein-level readout could substan­tially improve mRNA-LNP screening and accelerate the development of new nu­cleic-acid-based therapeutic and vaccine candidates.

Newer techniques, such as protein barcoding, are now being employed to ac­celerate and scale up this process. This multiplexed approach enables the simul­taneous evaluation of mRNA-LNP formu­lations in a single experiment, significantly accelerating the identification of lead can­didates with optimal biodistribution and transfection profiles, at a reduced cost.

THE BASICS OF PROTEIN BARCODING

Protein barcodes are information-rich short stretches of amino acids that can be genetically added to the coding sequences of proteins. Proteins expressing unique barcodes can be co-expressed together, and desired proteins can be selected for protein engineering, mRNA translation, therapeutic delivery mechanisms, and many other protein-screening applica­tions. Upon protein selection, distinct bar­codes associated with each expressed protein can be directly identified and quantified with single-molecule resolution via next-gen protein sequencing (NGPS). This approach has the potential to trans­form drug discovery and development, similar to how DNA barcodes have ad­vanced the field of genomics. Importantly, NGPS offers significant advantages over the use of MS for decoding protein bar­codes. These advantages include a simple, user-friendly workflow on a benchtop in­strument that distinguishes peptides based on recognition of specific amino acids rather than mass/charge ratio. Thus, pro­tein barcodes for NGPS readout can be engineered to have highly similar physical properties while remaining distinguishable by their amino acid sequences.

By appending the coding sequences for protein barcodes to each mRNA coding sequence, expressed proteins will have unique tags, despite the proteins them­selves having the same amino acid se­quence. Sequencing the barcodes on the Platinum Pro® NGPS platform allows rapid identification of which mRNA had the highest expression, resulting in the most abundant protein. These sequencing re­sults can also be used to determine the ef­fectiveness of different lipid nanoparticle delivery systems.

 ADVANTAGES FOR IN VIVO MRNA-LNP

While in vitro assays are essential for early screening, they can’t capture the full biological complexity of how mRNA-LNPs behave in the body. Once administered in vivo, LNPs interact with endogenous pro­teins that can coat the particle and signif­icantly influence biodistribution, an effect that is both unpredictable and impossible to replicate in vitro. In vivo studies are therefore critical to truly understand deliv­ery efficiency and tissue targeting. Protein barcoding offers a powerful solution by enabling multiplex-pooled screens with a direct protein-level readout, increasing the scale of screening while significantly re­ducing costs and the number of animals required for the all-important in vivo stud­ies (Figure 2).

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SIGNIFICANT REDUCTION IN IN VIVO MRNA-LNP SCREENING COSTS

As summarized in Table 1, a simu­lated study using multiplexing study with barcodes offers the potential to signifi­cantly improve the process economics of mRNA-LNP screening in animal models. By allowing multiple candidates to be tested simultaneously in a single animal, this approach maximizes data output per study and minimizes the number of ani­mals needed. The result is a more efficient screening process that reduces both time and development costs, accelerating the path to identifying optimal delivery vehi­cles, and aligns with recent FDA plans to reduce animal testing in drug develop­ment.⁹

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In vivo studies in mice using barcodes and NGPS can reduce costs by 71% and the number of animals required by a fac­tor of eight. In studies with non-human pri­mates (NHPs), the number of animals required can also be reduced eightfold, with an 86% reduction in costs.

PROTEIN BARCODES HELP UNLOCK THE FUTURE OF NUCLEIC ACID THERAPEUTICS

As mRNA and other nucleic-acid-based therapeutics and vaccines expand beyond liver-targeted applications, the need for precise, tissue-specific delivery becomes increasingly urgent. Protein bar­coding offers a transformative leap for­ward in this effort, one that not only addresses the limitations of current screen­ing technologies but also redefines the scale and efficiency of in vivo testing. By enabling multiplexed, protein-level read­outs within a single animal, this approach dramatically reduces both time and cost while improving the quality of data gener­ated.

More than just a technical advance­ment, protein barcoding is a catalyst for unlocking the full therapeutic potential of nucleic acid-based medicines. It empowers researchers to rapidly identify the most promising delivery systems, paving the way for safer, more effective treatments that reach beyond the liver and into a broader range of diseases and tissues. In short, protein barcoding doesn’t just streamline the development pipeline — it also opens new doors for innovation in ge­netic medicine.

*Cost estimates provided are for informational purposes only and are based on general as­sumptions regarding experimental design, throughput, and resource utilization. Actual sav­ings will vary depending on multiple factors, in­cluding but not limited to: the specific workflow and protocols used, the scale and frequency of experiments, reagent and consumable costs, labor costs, institutional pricing and discount structures, equipment depreciation, and regula­tory or compliance requirements. Quantum-Si makes no guarantees regarding specific cost re­ductions or financial outcomes. Customers should perform their own cost analysis based on their unique operational parameters.

 REFERENCES

1. Kulkarni, J.A., Witzigmann, D., Thomson, S.B., Chen, S., Leavitt, B.R., Cullis, P.R., and Van der Meel, R. (2021). The current landscape of nu­cleic acid therapeutics. Nat. Nanotechnol 16, 630–643. doi.org/10.1038/ s41565-021-00898-0
2. Khirallah, J., and Xu, Q. (2023). A high-throughput approach of screening nanoparticles for targeted delivery of mRNA. Cell Reports Methods 3, 100572. doi. org/10.1016/j.crmeth.2023.100572.
3. Guimaraes, P.P.G., Zhang, R., Spektor, R., Tan, M., Chung, A., Billings­ley, M.M., El-Mayta, R., Riley, R.S., Wang, L., Wilson, J.M., and Mitchell, M.J. (2019). Ionizable lipid nanoparticles encapsulating barcoded mRNA for accelerated in vivo delivery screening. J Control Release 316, 404–417. doi.org/10.1016/j.jconrel.2019.10.028
4. Cui, L., Pereira, S., Sonzini, S., van Pelt, S., Romanelli, S.M., Liang, L., Ulkoski, D., Krishnamurthy, V.R., Brannigan, E., Brankin, C., and Desai, A.S. (2022). Development of a high-throughput platform for screening lipid nanoparticles for mRNA delivery. Nanoscale 14, 1480- 1491. doi.org/10.1039/D1NR06858J.
5. Gu, L., Li, C., Aach, J., Hill, D.E., Vidal, M., and Church, G.M. (2014). Multiplex single-molecule interaction profiling of DNA-barcoded pro­teins. Nature 515, 554– 557. doi.org/10.1038/nature13761.
6. Biggs, B.W., Price, M.N., Lai, D., Escobedo, J., Fortanel, Y., Huang, Y.Y., Kim, K., Trotter, V.V., Kuehl, J.V., Lui, and L.M. (2024). High-throughput protein characterization by complementation using DNA barcoded fragment libraries. Mol Syst Biol 20, 1207–1229. doi:10.1038/ s44320-024-00068-z.
7. Suzuki, Y., Miyazaki, T., Muto, H., Kubara, K., Mukai, Y., Watari, R., Sato, S., Kondo, K., Tsukumo, S.I., Yasutomo, K., Ito, M., and Tsuka­hara, K. (2022). Design and lyophilization of lipid nanoparticles for mRNA vaccine and its robust immune response in mice and nonhuman primates. Mol Ther Nucleic Acids 30, 226-240. doi: 10.1016/j. omtn.2022.09.017.
8. Lowenthal, M.S., Antonishek, A.S., and Phinney, K.W., (2024). Quantifi­cation of mRNA in Lipid Nanoparticles Using Mass Spectrometry. Ana­lytical Chemistry 96, 1214- 1222. doi.org/10.1021/acs.analchem.3c04406.
9. https://www.fda.gov/news-events/press-announcements/fda-announces-plan-phase-out-animal-testing-requirement-monoclonal-antibodies-and-other-drugs.

Dr. Meredith Carpenter is Head of Scientific Affairs at Quantum-Si, where she manages external collaborations and publication strategy. Dr. Carpenter has over 10 years of experience in developing and deploying novel genomics and multi-omics tools in the biotech industry. Prior to Quantum-Si, she held roles as Director of Assay Development at Arc Bio and Senior Director of Strategic Alliances at Cantata Bio. She earned a BS in Biology from Emory University and a PhD in Molecular and Cell Biology from UC Berkeley, and she performed post-doctoral research in the Department of Genetics at Stanford University.