RNA therapeutics are changing the drug and medicine landscape. From the rapid development of the SARS-CoV-2 mRNA vaccine to clinical trials of RNA-based gene-editing, RNA therapeutics are quickly advancing. 

While these innovations are promising, developers must ensure their drug products meet regulatory standards. With IVT RNA, maintaining the integrity of the RNA is especially important to avoid impurities. Drug regulators are continuously improving their guidelines, and methods to check quality and integrity are evolving alongside these regulatory standards. 

Why IVT RNA integrity matters 

RNA is an inherently unstable molecule and will lose integrity over time. Temperature, pH, and length of the RNA all affect integrity, leading to denaturing or fragmentation under certain conditions. One prominent example is hydrolysis of the backbone, which can be exacerbated by pH changes or the presence of specific ions. In addition, RNase contamination can cleave RNA. The RNA must be a full, intact strand to make the correct protein for a therapeutic, so any breakage makes the therapeutic less effective.  

These factors are an issue for RNA therapeutics across the therapeutic lifecycle from development, to storage, to delivery. After a drug is developed, it must be stored at a high quality until it is administered, typically requiring the use of cold storage. Even after it is administered, the RNA must remain protected inside a delivery vehicle until it is taken up by cells and released into the cytoplasm. 

Standard methods to measure integrity 

Due to the critical importance of integrity in therapeutic efficacy, regulators have established strong guidelines for the amount of intact RNA that is required for release. Currently, most regulators recommend using well-established technologies such as capillary or agarose gel electrophoresis and liquid chromatography.  

One of the most common methods used to evaluate integrity is capillary gel electrophoresis. In this procedure, RNA is injected into a gel-filled capillary tube. RNA molecules then separate by size, with short fragments moving at different rates than full intact molecules. The size of the fragments present can be determined in comparison to a reference ladder. Although powerful, this approach is limited to only revealing the size of contaminating fragments. It can’t reveal the identity of the fragmented species or identify breakage hotspots. 

Sequencing for integrity 

Next-generation sequencing is an innovative technology that is increasingly used to gain insights into IVT RNA integrity and purity. For example, sequencing can answer regulatory questions about the 5’ cap and 3’ tail, determining the length of the cap and poly(A) tail. It can also identify the RNA’s sequence at a single-nucleotide resolution, including the identification of fragmented molecules. 

Nanopore sequencing is one approach that has been applied to identify fragmented RNAs. Since nanopore sequencing is long-read sequencing, it reads the whole RNA at once rather than fragmenting it as is done in short-read sequencing. This lets developers characterize the entire RNA, including the identification of specific fragments that are present as seen in work by Gunter et al. in Nature Communications.  

Integrity characterization at Eclipsebio 

At Eclipsebio, we offer innovative sequencing assays that help developers meet regulatory requirements. For example, eSENSE dsRNA locates dsRNA in an IVT RNA to show where IVT runoff is occurring along a sequence. This assay also measures how much dsRNA is present in an IVT RNA strand. When combined with an RNA structure identification assay, such as Eclipsebio’s eSHAPE assay, eSENSE dsRNA can help identify RNA secondary structures that are more prone to dsRNA formation, offering insights for developers to improve their therapeutic’s purity. 

Specifically for integrity, Eclipsebio’s nanopore sequencing approach uses Oxford Nanopore's technology to directly read an entire RNA sequence. This long-read sequencing can find the length of a poly(A) tail and profile an entire RNA transcript on a single-nucleotide level, allowing for investigations of RNA integrity. 

Using sequencing to achieve regulatory success 

As RNA therapeutics advance, so do regulatory requirements. Methods to measure IVT RNA quality such as eSENSE dsRNA and nanopore sequencing help drug developers keep up with changing regulatory guidelines and create stable, clean, and effective RNA therapeutics. 

Interested in how Eclipsebio can help you validate the integrity of your IVT RNA? Reach out to us to get started. 

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