In recent years, in vitro transcribed messenger RNA (mRNA) has emerged as a potential therapeutic platform. To fulfill its promise, effective delivery of mRNA to specific cell types and tissues needs to be achieved. Lipid nanoparticles (LNPs) are efficient carriers for short-interfering RNAs and have entered clinical trials. However, little is known about the potential of LNPs to deliver mRNA. Here, we generated mRNA-LNPs by incorporating HPLC purified, 1-methylpseudouridine-containing mRNA comprising codon-optimized firefly luciferase into stable LNPs. Mice were injected with 0.005–0.250 mg/kg doses of mRNA-LNPs by 6 different routes and high levels of protein translation could be measured using in vivo imaging. Subcutaneous, intramuscular and intradermal injection of the LNP-encapsulated mRNA translated locally at the site of injection for up to 10 days. For several days, high levels of protein production could be achieved in the lung from the intratracheal administration of mRNA. Intravenous and intraperitoneal and to a lesser extent intramuscular and intratracheal deliveries led to trafficking of mRNA-LNPs systemically resulting in active translation of the mRNA in the liver for 1–4 days. Our results demonstrate that LNPs are appropriate carriers for mRNA in vivo and have the potential to become valuable tools for delivering mRNA encoding therapeutic proteins.
Graphical representation of experimental approaches and data acquisition. Methods for the delivery and analysis of data are described in the Materials and Methods and Results sections.
mRNA-based therapy was first described in 1992 with the injection of vasopressin-encoding mRNA into rats with diabetes insipidus1, but underwent little development other than as an immunogen delivery vector until its immunogenicity and stability were addressed2. mRNA-based therapy has several conceptual advantages over other nucleic acid-based approaches. First, mRNA cannot integrate into the host genome, so there is no potential danger for insertional mutagenesis. Second, mRNA only requires cytosolic delivery in the host cell where it is translated into functional protein. Third, mRNA is translated transiently in cells and is degraded in a relatively short but controllable amount of time.
In order to use mRNA therapeutically, four main hurdles need to be overcome, 1) poor translatability, 2) lack of RNA stability, 3) inefficient in vivo delivery, and 4) its activation of innate immune sensors. To increase mRNA translatability and stability, 5′ cap, optimized 5′- and 3′-UTRs, and coding sequence and poly(A)-tail modifications can be introduced into the molecule3,4. Incorporation of modified nucleosides, such as pseudouridine or 1-methylpseudouridine and HPLC purification of the in vitro transcribed mRNA further increase protein translation and make mRNA immunologically silent2. The use of non-immunogenic mRNA is crucial, because a series of innate immune receptors (TLR3, TLR7, TLR8, RIG-I, MDA5, NOD2, PKR and others) recognize RNA resulting in the release of type I interferons and activation of interferon-inducible genes and inhibition of translation5.
Efficient delivery of mRNA into target cells in vivo is a major challenge. A variety of formulations have been developed to protect the nucleic acid from RNases and facilitate its uptake into cells. Positively charged lipids, cationic polypeptides, polymers, micelles or dendrimers have been used for in vivo RNA delivery, some formulations have already entered clinical trials in the field of cancer immunotherapy6.
There has been significant progress in overcoming many of the difficulties associated with in vivo delivery of mRNA and lipid nanoparticles (LNPs), containing ionizable cationic lipids, represent one of the most advanced technological platforms. The LNPs used in this study are 70 to 100 nm particles, prepared using an ionizable amino lipid, phospholipid, cholesterol and a PEGylated lipid, similar in composition to the LNPs that have recently proven to be safe and efficient tools for siRNA delivery7,8. Robust gene silencing has been demonstrated in several species including non-human primates9,10 and LNPs with encapsulated siRNAs have been successfully tested in human clinical trials. However, our knowledge is limited concerning mRNA delivery by LNPs. Geall and colleagues have recently developed an RNA self-replicating vaccine formulated in LNPs or a nanoemulsion and found that an intramuscular injection with very low doses of RNA induced protective immune responses against respiratory syncytial and influenza virus in mice11,12 and against respiratory syncytial virus and CMV in non-human primates13.
In the present study, LNP-encapsulated, HPLC-purified, 1-methylpseudouridine-containing mRNA encoding firefly luciferase was delivered into cultured cells and mice. Mice were injected with 0.1 μg (0.005 mg/kg), 1 μg (0.050 mg/kg), and 5 μg (0.250 mg/kg) of mRNA-LNP formulations by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, and intratracheal. A series of comparative studies were carried out investigating the levels and duration of mRNA translation into the encoded protein. We have found that administration of mRNA-LNP complexes results in large amounts of protein production in vivo for varying lengths of time demonstrating that LNPs are suitable tools for highly efficient mRNA delivery.
2. Materials and methods
2.1. Ethics statement
Primary human cells utilized in these experiments were isolated from leukopheresis samples obtained from healthy volunteers. Written informed consent using a protocol approved by the University of Pennsylvania Institutional Review Board was used.
The investigators faithfully adhered to the “Guide for the Care and Use of Laboratory Animals” by the Committee on Care of Laboratory Animal Resources Commission on Life Sciences, National Research Council. The animal facilities at the University of Pennsylvania are fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). All studies were conducted using protocols approved by the University of Pennsylvania IACUC.
Human embryonic kidney (HEK) 293T cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L–glutamine (Life Technologies) and 10% fetal calf serum (FCS) (HyClone) (complete medium). Immature human dendritic cells (DCs) were generated from elutriated monocytes cultured in RPMI (Life Technologies), containing glutamine (2 mM), 10% FCS, human IL-4 (100 ng/ml) and human GM-CSF (50 ng/ml) (R&D). Cells were maintained with fresh medium containing IL-4 and GM-CSF every 3 days and used on day 7.
2.3. mRNA production
mRNAs were produced as previously described14 using linearized plasmids encoding codon-optimized firefly luciferase (pLuc19) and T7 RNA polymerase (Megascript, Ambion). mRNAs were transcribed to contain 130 nucleotide-long poly(A) tails. 1-methylpseudouridine-5′-triphosphate (TriLink) instead of UTP was used to generate modified nucleoside-containing mRNA. RNAs were capped using the m7G capping kit with 2′-O-methyltransferase (ScriptCap, CellScript) to obtain cap1. mRNA was purified by Fast Protein Liquid Chromatography (FPLC) (Akta Purifier, GE Healthcare) as described earlier15. All RNAs were analyzed by denaturing or native agarose gel electrophoresis and were stored frozen at −20°C.
2.4. Formulation of the mRNA
Lipofectin (Invitrogen) complexing was performed, as described previously2 using 0.8 μl of Lipofectin and 0.1 to 1.0 μg of mRNA per well of a 96-well plate. Complexing of mRNA to TransIT mRNA (TransIT) (Mirus Bio) was performed according to the manufacturer’s instructions combining 0.1 or 0.3 μg mRNA with TransIT reagents, TransIT mRNA (0.34 μl) and Boost (0.22 μl) in a final volume of 18 μl DMEM, which was added to a single 96-well of cells.
HPLC-purified 1-methylpseudouridine-containing firefly luciferase-encoding mRNA was encapsulated in LNPs using a self-assembly process in which an aqueous solution of mRNA at pH 4.0 is rapidly mixed with a solution of lipids dissolved in ethanol8. LNPs used in this study were similar in composition to those described previously7,8, which contain an ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG-lipid (50:10:38.5:1.5 mol/mol), encapsulated RNA-to-total lipid ratio of ~0.05 (wt/wt) and a diameter of ~80nm. mRNA-LNP formulations were stored at −80°C at a concentration of mRNA of ~1 μg/μl.
2.5. Cell transfections
For Lipofectin complexed mRNA, medium was removed and 47 μl of complexed mRNA was added to 5 × 104 HEK293T or DCs per well. Cells were incubated for 1 h and the Lipofectin-mRNA mixture was replaced with 200 μl complete medium. For TransIT complexed mRNA, 17 μl of complex was added to cells cultured in 183 μl complete medium. mRNA-LNPs were preincubated with 0.1 μg human recombinant ApoE3 (Sigma) protein in 6 μl AIM V medium (Life Technologies) for 5 minutes at 37 °C or not. After preincubation, mRNA-LNPs were added to the cells cultured in 194 μl AIM V medium. Cells were lysed in firefly-specific lysis reagent (Promega) at 18 h or at the indicated times post mRNA addition. Aliquots were assayed for enzyme activity using the firefly luciferase assay system (Promega) and a MiniLumat LB 9506 luminometer (Berthold/EG&G; Wallac).
2.6. Administration of LNPs to mice
Female BALB/c mice aged 6 weeks were purchased from Harlan Laboratories. Increasing amounts (0.1 μg, 1.0 μg or 5.0 μg) of mRNA-LNPs in Dulbecco’s Phosphate Buffered Saline (PBS) were injected into animals intradermally (30 μl), intraperitoneally (200 μl), subcutaneously (200 μl), intramuscularly (30 μl) and intravenously (100 μl) with 3/10cc insulin syringes (BD Biosciences) using standard techniques16. A Penn-Century Microsprayer Aerosolizer (Model IA-1C) and FMJ-250 high pressure syringe was used for intratracheal delivery of mRNA-LNPs via intubation17.
2.7. Bioluminescence imaging studies
Bioluminescence imaging was performed with an IVIS Spectrum imaging system (Caliper Life Sciences). Mice were administered D-luciferin (Regis Technologies) at a dose of 150 mg/kg intraperitoneally. Mice were anesthetized after receiving D-luciferin in a chamber with 3% isoflurane (Piramal Healthcare Limited) and placed on the imaging platform while being maintained on 2% isoflurane via a nose cone. Mice were imaged at 5 minutes post administration of D-luciferin using an exposure time of 5 seconds or longer to ensure that the signal acquired was within effective detection range (above noise levels and below CCD saturation limit). Bioluminescence values were quantified by measuring photon flux (photons/second) in the region of interest where bioluminescence signal emanated using the Living IMAGE Software provided by Caliper.
2.8. Mathematical and statistical analyses
The half-life of protein translation by the delivered mRNA was calculated using the equation: t1/2=Δt X ln(2)/ln(N0/Nt), where Δt is the time between measurements, N0 is the starting value and Nt is the value at the end of the time being evaluated. Time intervals where saturation was evident and where translation was ending, low values were not calculated to exclude the effect of protein half-life. Area under the curve calculations were performed using the equation: AUC=Σ ti+1 − ti)/2 X (Ci + Ci+1, where ti is the starting time point, ti+1 is the finishing time point, Ci is the starting value and Ci+1 is the finishing value for each measurement over time. Means, standard error of the means, and student’s paired t-tests were determined using Microsoft Excel Software.
3.1. Transfection of HEK293T cells and primary human DCs with luciferase mRNA formulated with lipofectin, TransIT or LNPs
Immortalized cell lines and primary cells are barely transfectable with naked mRNA but 60 to 95 percent transfection efficiencies can be obtained when mRNA is complexed with cationic polymer or lipid-based reagents, such as TransIT or Lipofectin18. In the first set of experiments, HEK293T cells and human monocyte-derived dendritic cells (hDCs) were transfected with luciferase encoding nucleoside-modified mRNA complexed with TransIT and Lipofectin or encapsulated into LNPs (Fig. 1A). Notably, cells were exposed to Lipofectin-mRNA complexes for an hour in serum-free medium whereas cells were incubated with TransIT-mRNA and LNP-mRNA complexes in complete medium. In HEK293T cells, translation levels were slightly higher when LNPs delivered the mRNA compared to cationic polymer/lipid or lipid (Fig. 1A). On the contrary, in DCs, the level of luciferase was lower when LNPs were used for formulation of the mRNA as compared to using the commercially available reagents. Unlike the standard transfection reagents named above, the LNPs exhibit a net neutral surface charge at physiological pH, which results in maximal delivery to hepatocytes in vivo7, but the absence of a positive surface charge reduces cell uptake in vitro. In vivo, ionizable LNP containing nucleic acid therapeutics bind ApoE and are taken up by hepatocytes through receptor mediated endocytosis19,20. Incubation of mRNA-LNPs with recombinant ApoE protein prior to transfection of DCs significantly increased uptake and subsequent translation of the luciferase encoding mRNA (Fig. 1A). HEK293T cells are decorated with ApoE receptor and produce its ligand as well21, hence, not surprisingly, addition of exogenous ApoE did not result in elevated luciferase production in this cell line (Fig. 1A).
The kinetics and dose response of protein translation were examined in HEK293T cells transfected with increasing amounts of Luc mRNA formulated with cationic polymer and/or lipid or LNPs. Transfecting higher amounts of cationic polymer and/or lipid-complexed Luc mRNA resulted in higher protein levels at 12 and 24 hours post transfection (Fig. 1B). Interestingly, LNP-formulated mRNA translated most efficiently at 24 hours post transfection regardless of the amount of mRNA transfected into cells (Fig. 1B). Most likely saturation of the translational or uptake capacity of the cells occurred at the lowest dose of delivered mRNA.
3.2. mRNA-LNP administration into mice
To examine the duration and distribution of protein production from mRNA-LNPs in vivo, 5.0 μg luciferase mRNA-LNPs were administered into mice using commonly applied therapeutic delivery routes and protein translation was followed over time. Intravenous (retro-orbital) and intraperitoneal delivery resulted in mRNA-LNP trafficking to the liver (Fig. 2), which is in accordance with previous observations for siRNA10,19. In the liver, mRNA was translated to functional protein and very strong bioluminescent signal was measured in the first 24 hours post injection. Translation abated relatively quickly in the liver and there was no measurable luciferase activity at 3 days after mRNA-LNP injection. A small fraction of activity remained at the site of the injections (around the eyes and in the abdomen) and continued translation was measured for up to 7 days demonstrating the rapid turnover of translatable mRNA in the liver.