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Metabolically stabilized double-stranded mRNA polyplexes.

Jacob A Poliskey1, Samuel T Crowley1, Raghu Ramanathan1

  • 1Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA, 52242, USA.

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Researchers developed double-stranded mRNA (dsmRNA) to overcome mRNA instability for gene therapy. This novel dsmRNA form exhibits significantly enhanced metabolic stability and potent transfection efficiency in vivo.

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Area of Science:

  • Molecular Biology
  • Gene Therapy
  • Biochemistry

Background:

  • Messenger RNA (mRNA) exhibits metabolic instability, limiting its application in gene therapy due to rapid RNase digestion.
  • Plasmid DNA is more stable than mRNA in circulation after systemic administration.

Purpose of the Study:

  • To enhance the metabolic stability and transfection efficiency of mRNA for gene therapy applications.
  • To investigate the potential of double-stranded mRNA (dsmRNA) as a stabilized mRNA platform.

Main Methods:

  • Hybridization of complementary reverse mRNA with forward mRNA to create double-stranded mRNA (dsmRNA).
  • RNase A digestion assays to compare metabolic stability of dsmRNA and single-stranded mRNA (ssmRNA).
  • Formulation of dsmRNA polyplexes using a PEG-peptide for enhanced stability and delivery.
  • Hydrodynamic dosing in mice followed by bioluminescence imaging to assess liver transfection efficiency.

Main Results:

  • dsmRNA demonstrated a 3000-fold increase in metabolic stability compared to ssmRNA.
  • Formulated dsmRNA polyplexes showed an additional 3000-fold stability improvement.
  • dsmRNA polyplexes exhibited potent transfection efficiency in mouse liver following hydrodynamic dosing.
  • Hybridization affecting UTRs led to a tenfold decrease in expression; repeat dosing showed transient expression without apparent immune response.
  • dsmRNA did not function as a Dicer substrate, as indicated by failed knockdown experiments.

Conclusions:

  • Double-stranded mRNA (dsmRNA) represents a novel, metabolically stable, and transfection-competent alternative to single-stranded mRNA for gene therapy.
  • Maximal stability was achieved with fully complementary dsmRNA polyplexes, highlighting their potential for in vivo applications.
  • The findings establish dsmRNA as a promising platform for overcoming mRNA delivery challenges.