The realm of druggable targets has expanded significantly with the advent of mRNA therapeutics. Scientists require exceptional accuracy and confidence to adequately characterize these therapeutics and their anticipated effects. To this end, Droplet Digital PCR (ddPCR) technology has proven invaluable. With unparalleled precision and absolute quantification, Droplet Digital PCR is revolutionizing the field of mRNA therapeutic development and manufacturing.

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The range of druggable targets has been greatly expanded by the introduction of mRNA-based therapeutics, which can target the template of any protein, peptide, or protein fragment. Additionally, because RNA serves as the intermediary between the DNA template and the effector protein, it is suitable for a wide range of therapeutic applications, from targeting genetic disorders to vaccine development (Qin et al. 2022). These therapeutics either provide or destroy RNA targets to achieve a therapeutic effect. For instance, mRNA-based vaccines provide an mRNA template that rapidly stimulates an immune response against pathogens or cancer cells (Pardi et al. 2018).

The development of efficacious mRNA-based therapeutics comes with unique challenges. Ensuring a drug’s potency, maintaining RNA integrity, and accurately characterizing the length of an RNA product’s poly(A) tail are all critical, making precise quantification of RNA components and targets essential to drug production. While traditional quantitative PCR (qPCR) is a common approach, it cannot achieve absolute quantification, potentially introducing variability into the developmental pipeline by requiring a new standard curve for each run.

ddPCR technology offers a solution for achieving the precise RNA quantification required for mRNA therapeutic development. The assay partitions samples into 20,000 individual droplets, each undergoing a separate PCR reaction, enabling absolute quantification and unparalleled precision. With the exceptional ability to detect even low levels of RNA, ddPCR assays are the optimal choice for mRNA therapeutic development and manufacturing. In this article, we will describe the benefits of ddPCR technology, highlighting examples where ddPCR workflows have revolutionized mRNA therapeutic development.

Precise Dosing Requires Absolute Quantification

Accurate RNA quantification is crucial to the success of RNA-based therapies. Because the RNA molecule itself is the “drug” in these therapeutic products, absolute quantification is essential for establishing safety and determining effective dosing. Any errors could lead to significant losses in time, resources, and public confidence. Moreover, researchers need to calculate the precise change in RNA expression observed in a patient to determine whether a treatment performs as expected. Absolute precision is essential at every stage of RNA therapeutic development.

Endpoint Assays Take the Guesswork Out of Confirming RNA Integrity

ddPCR assays offer unparalleled precision due to sample partitioning, with each droplet undergoing a single discrete PCR reaction. This unique approach eliminates the requirement for standard curves and enables researchers to achieve absolute RNA quantification, facilitating accurate dose determination, increasing reproducibility, and saving time throughout development.

Another important benefit of ddPCR assays is their ability to distinguish intact nucleic acid sequences to confirm their integrity, a key element of making safe and effective RNA therapeutics. While traditional PCR-based assays can detect the presence of short RNA sequences, they cannot indicate whether these sequences are derived from larger, intact molecules or degraded nucleic acids. Using ddPCR assays, however, researchers can get a clear, binary indication of whether intact RNA sequences are present, allowing for a better assessment of therapeutic RNA integrity.  For example, research by Mello and colleagues (preprint: Mello et al. 2020) on the degradation of SARS-CoV-2 RNA demonstrated how the partitioning in ddPCR technology enabled the researchers to detect the presence of an intact viral genome by determining whether specific sequences were present on the same larger nucleic acid.

Measuring Poly(A) Tail Length With ddPCR Assays

In addition to its conventional uses for nucleic acid detection, ddPCR technology can be used to measure poly(A) tail length. Poly(A) tails of 150-250 nucleotides are present on nearly all endogenous mRNAs and are vital for the mRNA maturation, stability, and trafficking. Deadenylation, or removal of the poly(A) tail, can initiate mRNA degradation (Liu et al. 2022). RNA therapeutics and vaccines require a poly(A) tail to protect the product from RNA degradation and ensure endogenous translation.

When developing the COVID-19 mRNA–based vaccine, Pfizer/BioNTech utilized ddPCR assays to verify the integrity of their therapeutic product’s poly(A) tail (Daniel et al. 2022). The widespread success of the Pfizer/BioNTech vaccine — and the unprecedented timeframe in which the vaccine was developed — demonstrates the importance of ddPCR technology in quantifying small but essential components of mRNA therapeutics.

Realizing the Full Potential of RNA Therapeutics

Since its inception, ddPCR technology has been the go-to method for quantifying nucleic acids. mRNA-based therapies present unique development and deployment challenges, such as accurately quantifying and ensuring the integrity of therapeutic mRNA. Industry leaders can address these hurdles with the unmatched sensitivity and precision of ddPCR technology, which is revolutionizing the development of mRNA therapeutics. Whether detecting rare contaminants, quantifying RNA-based drugs, or measuring therapeutic effects on host RNA levels, ddPCR is a game changer at every stage of mRNA therapeutics development.

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References

Daniel S et al. (2022). Quality by Design for enabling RNA platform production processes. Trends Biotechnol 40, 1213–1228.

Liu J et al. (2022). Molecular insights into mRNA polyadenylation and deadenylation. Int J Mol Sci 23, 10985.

Mello C et al. (2020). Absolute quantification and degradation evaluation of SARS-CoV-2 RNA by droplet digital PCR. medRxiv. Preprint. https://doi.org/10.1101/2020.06.24.20139584, accessed April 10, 2023.

Pardi N et al. (2018). mRNA vaccines — a new era in vaccinology. Nat Rev Drug Discov 17, 261–279.

Qin S et al. (2022). mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther 7, 166.

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