Advancing Gene Therapy: Enzyme Selection For Effective RNA Oligonucleotide Mapping

New gene therapy modalities, such as CRISPR guide RNA (single guide ribonucleic acid [sgRNA]) and messenger RNA (mRNA), continue to make progress in both primate and first-in-human trials. As this progress builds, the industry remains accountable for characterizing these molecules to meet the requirements of regulatory authorities. The sequence of ribonucleic acid chains is a critical quality attribute (CQA) as it significantly influences the drug’s efficacy. Moreover, in the case of CRISPR molecules, any ambiguity in sequence can also translate to undesired off-target effects. Similarly to peptide mapping in therapeutic protein characterization, mRNA oligonucleotide mapping by mass spectrometry (MS) is the preferred method because of its ease of implementation in regulated environments when compared to next-generation sequencing (NGS). One of the aims of this study was to make oligonucleotide mapping as accessible as peptide mapping. Oligonucleotide mapping gives a direct molecular compositional analysis of drug substance components. It also has the potential to support multi-attribute monitoring (MAM) approaches, such as 5’-capping determination and 3’ poly(A) tail length distribution in mRNA molecules, as well as the pinpoint confirmation of important modifications incorporated into CRISPR guides. In this study, four enzymes were evaluated—RNase T1, RNase 4, Cusativin, and MC1 —for oligo mapping of sgRNA and mRNA of varying lengths. The impact of missed cleavages on sequence coverage as determined by bioinformatics tools will be highlighted, as well as the effect of shorter oligos on sequence coverage.