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Efficient expression of stabilized mRNA PEG-peptide polyplexes in liver
S T Crowley1, J A Poliskey1, N J Baumhover1
1Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA, USA.
Gene Therapy
|July 1, 2015
Summary
Optimizing messenger RNA (mRNA) with human beta-globin untranslated regions (UTRs) and a novel PEG-peptide significantly boosted liver gene expression. This advancement offers a promising nonviral vector for targeted liver cell transfection.
Area of Science:
- Biotechnology
- Molecular Biology
- Gene Therapy
Background:
- In vitro transcribed mRNA offers potential for gene therapy but faces challenges in expression efficiency and stability.
- Hydrodynamic delivery is a method for introducing nucleic acids into the liver, but efficiency can be limited.
- Untranslated regions (UTRs) and delivery vectors play crucial roles in mRNA expression and stability.
Purpose of the Study:
- To significantly enhance the expression efficiency of in vitro transcribed mRNA in the liver.
- To develop and evaluate a novel nonviral vector for mRNA delivery to hepatocytes.
- To investigate the protective effects of a novel peptide against RNase degradation.
Main Methods:
- Codon optimization of mRNA with flanking 3' and 5' human beta-globin untranslated regions (UTRs).
- Formation of nanoparticle UTR mRNA polyplexes using a novel polyacridine polyethylene glycol (PEG) peptide.
- Hydrodynamic delivery of constructs into the liver for expression analysis.
- Assessment of expression efficiency and stability through luciferase assays and serum incubation.
Main Results:
- A 2000-fold increase in liver expression efficiency was achieved with codon-optimized UTR mRNA compared to unoptimized mRNA.
- The novel polyacridine PEG-peptide resulted in an additional 15-fold increase in expression efficiency.
- Combined UTR mRNA PEG-peptide polyplexes demonstrated a 3500-fold increase in expression over unmodified mRNA.
- UTR mRNA polyplexes showed 10-fold greater expression than an equivalent dose of pGL3 DNA.
- Maximal expression was sustained between 4 and 24 hours post-delivery.
- The PEG-peptide protected UTR mRNA polyplexes from RNase degradation by binding to double-stranded regions.
Conclusions:
- Codon-optimized UTR mRNA, when formulated into PEG-peptide polyplexes, represents a highly efficient nonviral vector for liver gene delivery.
- This approach significantly enhances mRNA expression efficiency and stability, overcoming previous limitations.
- The developed system circumvents the need for nuclear uptake, offering a direct pathway for hepatocyte transfection.
- These findings represent a significant advancement toward targeted gene delivery systems for liver diseases.
