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Reverse-phase chromatography removes double-stranded RNA, fragments, and residual template to decrease immunogenicity

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Molecular Therapy. Nucleic Acids
|April 1, 2025
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Summary

Purifying messenger RNA (mRNA) with ion-pair reverse-phase (IP-RP) chromatography effectively removes impurities like double-stranded RNA (dsRNA). This enhances protein translation and reduces innate immune responses for improved mRNA therapeutics.

Keywords:
HPLCMT: Delivery StrategieschromatographydsRNAmRNAsaRNAvaccines

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Biochemistry

Background:

  • Messenger RNA (mRNA) production via in vitro transcription generates impurities, notably double-stranded RNA (dsRNA).
  • dsRNA activates innate immune responses, inhibiting protein synthesis and reducing mRNA therapeutic efficacy.
  • Current mRNA purification methods may not fully remove all critical impurities like dsRNA, hybridized fragments, and DNA templates.

Purpose of the Study:

  • To comprehensively study ion-pair reverse-phase (IP-RP) liquid chromatography (LC) purification on monolith supports for mRNA polishing.
  • To demonstrate the removal of dsRNA, hybridized RNA fragments, and residual DNA template using IP-RP LC.
  • To evaluate the impact of this purification method on mRNA and self-amplifying RNA (saRNA) quality and function.

Main Methods:

  • Development of a step elution methodology for IP-RP LC purification on monolith supports.
  • Application of the method at microgram scale using novel spin columns operated by centrifugation.
  • Assessment of SDVB (a type of monolith support) efficiency across various molecular sizes and investigation of temperature control for dsRNA removal from saRNA.

Main Results:

  • IP-RP LC effectively removed dsRNA, hybridized RNA fragments, and residual DNA template, surpassing standard mRNA capture methods.
  • SDVB-purified mRNA and saRNA demonstrated significantly increased transgene expression in cell-based assays.
  • Purified mRNA and saRNA showed reduced activation of innate immunity in A549 cells at early time points.

Conclusions:

  • IP-RP purification on monolith supports is crucial for producing high-quality mRNA and saRNA.
  • This purification strategy simplifies technological requirements for clinical mRNA and saRNA manufacturing.
  • Effective impurity removal via IP-RP LC enhances mRNA translation efficiency and reduces immunogenicity.