Metabolically stabilized double-stranded mRNA polyplexes

Gene Therapy
J A PoliskeyKevin G Rice

Abstract

The metabolic instability of mRNA currently limits its utility for gene therapy. Compared to plasmid DNA, mRNA is significantly more susceptible to digestion by RNase in the circulation following systemic dosing. To increase mRNA metabolic stability, we hybridized a complementary reverse mRNA with forward mRNA to generate double-stranded mRNA (dsmRNA). RNase A digestion of dsmRNA established a 3000-fold improved metabolic stability compared to single-stranded mRNA (ssmRNA). Formulation of a dsmRNA polyplex using a PEG-peptide further improved the stability by 3000-fold. Hydrodynamic dosing and quantitative bioluminescence imaging of luciferase expression in the liver of mice established the potent transfection efficiency of dsmRNA and dsmRNA polyplexes. However, hybridization of the reverse mRNA against the 5' and 3' UTR of forward mRNA resulted in UTR denaturation and a tenfold loss in expression. Repeat dosing of dsmRNA polyplexes produced an equivalent transient expression, suggesting the lack of an immune response in mice. Co-administration of excess uncapped dsmRNA with a dsmRNA polyplex failed to knock down expression, suggesting that dsmRNA is not a Dicer substrate. Maximal circulatory stability was achieved using a full...Continue Reading

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Citations

Aug 28, 2020·Molecular Pharmaceutics·Satoshi UchidaHoracio Cabral
Sep 11, 2020·Analytical Biochemistry·Raghu RamanathanKevin G Rice
Dec 22, 2021·Molecular Pharmaceutics·Joachim Justad RøiseNiren Murthy

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Methods Mentioned

BETA
M13 sequencing
PCR
in vitro transcription
electrophoretic band mobility shift
electrophoresis
dynamic light scattering
bioluminescence imaging
transfection

Software Mentioned

GraphPad Prism
GraphPad
VisionWorks LS

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