Ethical statement
All experiments were performed in compliance with all relevant ethical regulations as approved by the Institutional Biosafety Committee (IBC) of the Broad Institute (protocol #IBC-2017-00146). All animal experiments were approved by the Institutional Animal Care and Use Committee of the Broad Institute (protocol ID 0017-09-14-2). Animal maintenance complied with all relevant ethical regulations and were consistent with local, state and federal regulations as applicable, including the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
Plasmid construction
G-blocks encoding the human α-globin 5′ untranslated region (UTR) and 3′ FI element were synthesized de novo by IDT. In vitro transcription vectors were cloned by inserting UTRs into a pET45 vector via Gibson assembly using Gibson Assembly Master Mix (E2611L, NEB) and transformation into chemically competent Stbl3 cells. A hard-coded A30LA70 polyA tail was added by PCR and ligation using the KLD enzyme mix (New England Biolabs). Subsequent coding sequences were inserted by digestion with NcoI and XhoI and Gibson assembly. Plasmid sequences were verified via next-generation sequencing, long-read sequencing (Primordium Labs) and PCR to verify the length of polyA tails.
In vitro transcription and LiCl purification of mRNA
Plasmids were linearized, and a T7-driven in vitro transcription reaction (Life Technologies) was performed to generate mRNA with 101 nucleotide long polyA tails. The 5′ UTR and the 3′ FI elements contained sequences from the human α-globin gene. Capping of mRNA was performed in concert with transcription through addition of a trinucleotide cap1 analogue CleanCap, and m1Ψ-5′-triphosphate (TriLink) was incorporated into the reaction instead of uridine-5′-triphosphate (UTP; Supplementary Fig. 1a). LiCl-based purification of mRNA was performed, mRNAs were then checked on an agarose gel and by a TapeStation RNA ScreenTape Analysis (Agilent; Supplementary Fig. 1b,c) before aliquoting at 1 µg µl−1 and storing at −80 °C.
DFI LNP production
We engineered a formulation of mRNA-encoded DFI within biodegradable lipopolyplexes, along with control formulations (Fig. 1e). To do this, we formulated LNPs by combining SM-102 as ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) in a molar ratio of 50:10:38.5:1.5. These were formulated into LNPs along with mRNA using microfluidic mixing using a NanoAssemblr Ignite nanoparticle formulation system (Cytiva). In brief, an ethanol phase containing the above formulated lipidoid, phospholipid, cholesterol and DMG-PEG master mix was mixed with an aqueous phase (10 mM citrate buffer, pH 3) containing mRNA at a flow rate ratio of 1:3 and at a lipidoid:RNA weight ratio of 10:1. Upon formulation, mRNA–LNPs were diluted in sterile NaCl and the buffer was exchanged by concentrating with a 30-kDa spin filter (UFC9030, MilliporeSigma) to replace residual ethanol. NaCl-diluted mRNA–LNPs were stored at 4 °C until use. For all subsequent experiments, we utilized these SM-102 mRNA–LNPs encapsulating m1Ψ-5′-triphosphate-modified and m7GpppNm-capped DFI, Luc or GFP mRNA, respectively.
LNP characterization
The hydrodynamic size, polydispersity index (PDI) and zeta potential (ZP) of LNPs were measured using a DynaPro NanoStar II (Wyatt). The mRNA encapsulation efficiency of LNPs were determined using a modified Quant-iT RiboGreen RNA assay (Invitrogen) and found to be more than 85% on average (Supplementary Fig. 1d). LNP endotoxin levels were consistently found to be less than 1 endotoxin unit per ml. The average hydrodynamic diameter was approximately 75 nm with a polydispersity index of 0.02–0.03 (Supplementary Fig. 1e).
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