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 β-globin untranslated regions (UTR mRNA) over an un-optimized mRNA without β-globin UTRs. Nanoparticle UTR mRNA polyplexes were formed using a novel polyacridine 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 µg dose of pGL3. Maximal expression was maintained from 4 to 24 hours. 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 non-viral vectors that circumvent the need for nuclear uptake, representing an advancement toward the development of a targeted gene delivery system to transfect liver hepatocytes.

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals

“…To date, naked, chemically modified, or protamine-complexed mRNA have shown promise in phase I/II cancer trials (24)(25)(26). Recently, preclinical development of materials specific for mRNA delivery has resulted in cationic polymers such as polymethacrylates (27)(28)(29), poly(aspartamides) (30,31), and polypeptides (32), as well as multicomponent cationic lipid or lipid-like formulations (21,(33)(34)(35)(36). In many of these examples, however, transfection efficiencies can be quite low, ranging 20-80% in cells (18), with likely much lower efficiencies in vivo, which requires either high mRNA doses or hydrodynamic injections (32,37).…”

Design concepts of polyplex micelles for in vivo therapeutic delivery of plasmid DNA and messenger RNA

“…34,35 However, nucleic acids loaded in carriers were degraded even in the absence of highly anionically charged molecules, for example, after incubation with serum or purified nuclease proteins, especially in the case of mRNA, which is highly susceptible to nuclease attack. 31,36,37 These results raise the possibility of nucleic acids degradation even without the complete release of nucleic acids from their carriers. PEG coating of polyplexes or lipoplexes is a promising strategy to prevent both of the nucleic acid degradation mechanisms described above, by inhibiting the attack of nucleases or polyanions on nucleic acids.…”

“…Heparan sulfate on liver sinusoids and glomerular basement membrane in particular possesses high anionic charge density and was shown to dissociate polyplexes and lipoplexes containing nucleic acids . However, nucleic acids loaded in carriers were degraded even in the absence of highly anionically charged molecules, for example, after incubation with serum or purified nuclease proteins, especially in the case of mRNA, which is highly susceptible to nuclease attack . These results raise the possibility of nucleic acids degradation even without the complete release of nucleic acids from their carriers.…”

Nanoscale platforms for messenger RNA delivery

“…Previous studies also investigated a number of polypeptides for mRNA delivery. For example, Crowley, Poliskey, Baumhover, and Rice (2015) installed acridine and PEG5k on the polylysine backbone peptide. The formulated luciferase mRNA polyplexes extended mRNA degradation time and led to a high luminescence signal in the liver after hydrodynamic injection in mice (Crowley et al, 2015).…”