Gene Therapy and Oligonucleotide Compendium - Volume 1

Importance of the Topic

The rapid expansion of gene and oligonucleotide therapeutics has created a critical need for precise, high-throughput analytical methods. Accurate characterization of viral vectors, plasmids, nucleic acids, and oligonucleotides is essential for ensuring product safety, efficacy, and regulatory compliance. Advanced techniques such as capillary electrophoresis with laser-induced fluorescence (CE-LIF), high-resolution mass spectrometry and capillary-based sequencing platforms have become pivotal for meeting these challenges.

Objectives and Study Overview

This compendium reviews state-of-the-art analytical workflows across six key areas:

• AAV capsid protein purity and empty/partial/full vector analysis
• Nucleic acid sizing of DNA and RNA over broad size ranges
• Plasmid topology analysis and degradation monitoring
• Selection of high-affinity aptamers by CE-SELEX
• Capillary-based DNA sequencing methods for large constructs
• SNP and mutation genotyping by single-base extension (SNPStart kit)

Methodologies and Instrumentation

All workflows leverage CE-based separation using the SCIEX PA 800 Plus or GeXP Genetic Analysis System, often interfaced with laser-induced fluorescence detection or coupled with high-resolution MS (TripleTOF, QTOF). Key reagents and kits include:

• SDS-MW and dsDNA 1000 kits with LIFluor EnhanCE dye for CE-LIF
• CE-SELEX methodology utilizing plain capillaries for aptamer selection
• DTCS and DTCS Quick-Start kits for capillary dye-terminator sequencing
• SNPStart single-base extension master mix for multiplex genotyping

Main Results and Discussion

AAV capsid analysis by CE-SDS and CE-LIF: baseline separation of VP1/VP2/VP3 with RSD <1%, LOD 1×10¹⁰ GC/mL; CE-IEF methods for full/empty vector quantification correlate with HPLC.
DNA sizing: CE-LIF achieved accurate sizing from 100 bp to 15 kb with precision ≤0.27 bp and LOD 0.79 ng/mL.
RNA analysis: CE-LIF separated 0.2 kb–6.5 kb RNA with LOQ 0.33 ng/mL and linearity R²>0.995; single-base resolution down to 15 nt for sgRNA and CRISPR components.
Plasmid purity: CE-LIF separated supercoiled, linear and open circular isoforms of 5–10 kb plasmids in <15 min with RSD <0.7%; suitable for stability studies.
CE-SELEX: Aptamers against IgE, NMM and other targets were isolated in 2–4 rounds; Kd values as low as 27 nM; aptamers showed >40× selectivity.
Capillary sequencing: GeXP system achieved >900 bp 98% accuracy cutoff, extended reads by >20% via optimized voltage, temperature, injection and preheat protocols; BAC end sequencing yielded ~800 bp average high-quality reads.
SNP genotyping: SNPStart kit enabled multiplexed (up to 10-plex) single-base extension with allele-balance ratio ~1:1.6 and >99% calling accuracy from cell line and whole-genome amplified DNA.

Benefits and Practical Applications

• Significantly shorter analysis times and higher throughput than slab gels or ultracentrifugation
• Improved sensitivity and reproducibility for critical quality attributes of gene therapy vectors and oligonucleotide drugs
• Flexible CE-based platforms accommodate diverse workflows from aptamer discovery to SNP screening
• Methods are broadly transferable across instruments and laboratories

Future Trends and Opportunities

Integration of CE-MS and microfluidics promises even higher sensitivity for low-abundance species. Automation of sample preparation (e.g., robotics, microdroplet systems) will further accelerate analysis. Advances in AI-driven data interpretation may streamline QC decision making and facilitate online monitoring during bioprocessing of gene therapies.

Conclusion

Capillary electrophoresis and related techniques represent a versatile toolbox for comprehensive characterization of gene and oligonucleotide therapeutics. By combining high resolution, speed, and adaptability, these methods address critical analytical challenges across development, manufacturing, and quality control of next-generation biopharmaceutical modalities.

Reference

1. FDA Points to Consider on Plasmid DNA Vaccines for Preventive Infectious Diseases. 1996. 96N-0400.
2. Quirino, J. (2015) Modern Injection Modes (Stacking) for CE. Wiley.
3. Mendonsa, S. D.; Bowser, M. T. In Vitro Evolution of Functional DNA using CE. J. Am. Chem. Soc. 2004, 126, 20–21.
4. Yang, J. B.; Bowser, M. T. CE-SELEX Selection of Catalytic DNA Aptamers. Anal. Chem. 2013, 85 (3), 1525–1530.
5. Wiegand, T. W.; et al. High Affinity DNA Ligands to Human IgE. J. Immunol. 1996, 157, 221–230.