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Ionizable Polymeric Micelles with Phenylalanine Moieties Enhance Intracellular Delivery of Self-Replicating RNA for

Lucas Mixich1, Eger Boonstra1, Keita Masuda1

  • 1Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8658, Japan.

Biomacromolecules
|January 5, 2024
PubMed

Insights

New self-replicating RNA (RepRNA) delivery systems using phenylalanine-modified micelles enhance protein expression. This breakthrough offers a promising alternative to traditional mRNA therapeutics, reducing dosage needs and improving treatment efficacy.

Area of Science:

  • Biotechnology
  • Nanomedicine
  • RNA Therapeutics

Background:

  • Messenger RNA (mRNA) therapeutics show promise but are limited by short half-lives and transient protein expression.
  • Self-replicating RNA (RepRNA) offers a strategy for enhanced protein production and reduced dosing requirements.
  • Efficient delivery systems are crucial for maximizing the therapeutic potential of RNA-based treatments.

Purpose of the Study:

  • To develop and characterize novel polymeric micelles for the efficient delivery of RepRNA.
  • To evaluate the stability, cellular uptake, and endosomal escape of RepRNA-loaded micelles.
  • To assess the in vitro and in vivo protein expression efficiency of the developed delivery system.

Main Methods:

  • Synthesis of poly(ethylene glycol)-poly(glycerol) (PEG-PG) block copolymers modified with phenylalanine (Phe) moieties.
  • Encapsulation of RepRNA into PEG-PG(Phe) micelles and characterization of micelle size and RepRNA integrity.
  • Assessment of micelle stability in serum and pH-responsive endosomal escape mechanism.
  • In vitro protein expression studies and in vivo evaluation of protein expression compared to commercial reagents.

Main Results:

  • Sub-100 nm polymeric micelles encapsulating RepRNA were successfully fabricated using Phe-modified PEG-PG copolymers.
  • The PEG-PG(Phe) micelles demonstrated excellent RepRNA stability in RNase-rich serum.
  • The micelles exhibited pH-responsive endosomal disruption, facilitating cytosolic RepRNA delivery.
  • In vitro studies showed strong protein expression, and in vivo studies demonstrated enhanced, long-lasting protein expression compared to controls.

Conclusions:

  • Phenylalanine-modified PEG-PG micelles provide an effective platform for delivering RepRNA.
  • This novel delivery system enhances RepRNA stability and promotes efficient cytosolic delivery.
  • The system achieves superior in vitro and in vivo protein expression, outperforming existing reagents and offering a promising advancement in RNA therapeutics.

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