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Efficient expression of stabilized mRNA PEG-peptide polyplexes in liver.

ABSTRACT: The expression efficiency in liver following hydrodynamic delivery of in vitro transcribed mRNA was improved 2000-fold using a codon-optimized mRNA luciferase construct with flanking 3' and 5' human ?-globin untranslated regions (UTR mRNA) over an unoptimized mRNA without ?-globin UTRs. Nanoparticle UTR mRNA polyplexes were formed using a novel polyacridine polyethylene glycol (PEG) peptide, resulting in an additional 15-fold increase in expression efficiency in the liver. The combined increase in expression for UTR mRNA PEG-peptide polyplexes was 3500-fold over mRNA lacking UTRs and PEG-peptide. The expression efficiency of UTR mRNA polyplex was 10-fold greater than the expression from an equivalent 1??g dose of pGL3. Maximal expression was maintained from 4 to 24?h. Serum incubation established the unique ability of the polyacridine PEG-peptide to protect UTR mRNA polyplexes from RNase metabolism by binding to double-stranded regions. UTR mRNA PEG-peptide polyplexes are efficient nonviral vectors that circumvent the need for a nuclear uptake, representing an advancement toward the development of a targeted gene delivery system to transfect liver hepatocytes.

SUBMITTER: Crowley ST

PROVIDER: S-EPMC4670273 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Efficient expression of stabilized mRNA PEG-peptide polyplexes in liver.

Crowley S T ST Poliskey J A JA Baumhover N J NJ Rice K G KG

Gene therapy 20150630 12

The expression efficiency in liver following hydrodynamic delivery of in vitro transcribed mRNA was improved 2000-fold using a codon-optimized mRNA luciferase construct with flanking 3' and 5' human β-globin untranslated regions (UTR mRNA) over an unoptimized mRNA without β-globin UTRs. Nanoparticle UTR mRNA polyplexes were formed using a novel polyacridine polyethylene glycol (PEG) peptide, resulting in an additional 15-fold increase in expression efficiency in the liver. The combined increase ...[more]

PMID: 26125604

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Project description:Nonviral gene therapy has high potential for safely promoting tissue restoration and for treating various genetic diseases. One current limitation is that conventional transfection reagents such as polyethylenimine (PEI) form electrostatically stabilized plasmid DNA (pDNA) polyplexes with poor colloidal stability. In this study, a library of poly(ethylene glycol-b-(dimethylaminoethyl methacrylate-co-butyl methacrylate)) [poly(EG-b-(DMAEMA-co-BMA))] polymers were synthesized and screened for improved colloidal stability and nucleic acid transfection following lyophilization. When added to pDNA in the appropriate pH buffer, the DMAEMA moieties initiate formation of electrostatic polyplexes that are internally stabilized by hydrophobic interactions of the core BMA blocks and sterically stabilized against aggregation by a PEG corona. The BMA content was varied from 0% to 60% in the second polymer block in order to optimally tune the balance of electrostatic and hydrophobic interactions in the polyplex core, and polymers with 40 and 50 mol% BMA achieved the highest transfection efficiency. Diblock copolymers were more stable than PEI in physiologic buffers. Consequently, diblock copolymer polyplexes aggregated more slowly and followed a reaction-limited colloidal aggregation model, while fast aggregation of PEI polyplexes was governed by a diffusion-limited model. Polymers with 40% BMA did not aggregate significantly after lyophilization and produced up to 20-fold higher transfection efficiency than PEI polyplexes both before and after lyophilization. Furthermore, poly(EG-b-(DMAEMA-co-BMA)) polyplexes exhibited pH-dependent membrane disruption in a red blood cell hemolysis assay and endosomal escape as observed by confocal microscopy.Lyophilized polyplexes made with the lead candidate diblock copolymer (40% BMA) also successfully transfected cells in vitro following incorporation into gas-foamed polymeric scaffolds. In summary, the enhanced colloidal stability, endosomal escape, and resultant high transfection efficiency of poly(EG-b-(DMAEMA-co-BMA))-pDNA polyplexes underscores their potential utility both for local delivery from scaffolds as well as systemic, intravenous delivery.

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Efficient expression of stabilized mRNA PEG-peptide polyplexes in liver.

Publications

Efficient expression of stabilized mRNA PEG-peptide polyplexes in liver.

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