mRNA Polyplexes with Post-Conjugated GALA Peptides Efficiently Target, Transfect, and Activate Antigen Presenting Cells

Abstract

Vaccines based on mRNA have emerged as potent systems to elicit CD8 T cell responses against various cancers and viral infectious diseases. The efficient intracellular delivery of mRNA molecules encoding antigens into the cytosol of antigen-presenting cells (APCs) is still challenging, requiring cell attachment, active uptake, and subsequent endosomal escape. Here, we report a facile approach for the formulation of peptide-functionalized mRNA polyplexes using copper-free click chemistry to promote presentation of mRNA antigen by dendritic cells (DCs). After screening different membrane active peptides, GALA modified mRNA polyplexes (PPx-GALA) with a size around 350 nm and with a slightly negative surface charge (-7 mV), exhibited the highest EGFP-mRNA transfection in RAW 246.7 macrophages (∼36%) and D1 dendritic cells (∼50%) as compared to polyplexes decorated with melittin or LEDE peptides. Interestingly, we found that PPx-GALA enters DCs through sialic acid mediated endo/phagocytosis, which was not influenced by DC maturation. The PPx-GALA formulation exhibited 18-fold higher cellular uptake compared to a lipofectamine mRNA formulation without inducing cytotoxicity. Live cell imaging showed that PPx-GALA that were taken up by endocytosis induced calcein release from endosomes into the cytosol. DCs treated with PPx-GALA containing mRNA encoding for OVA displayed enhanced T cell responses and DC maturation. Collectively, these data provide a strong rationale for further study of this PPx-GALA formulation in vivo as a promising mRNA vaccine platform.

mRNA Transfection-Induced Activation of Primary Human Monocytes and Macrophages: Dependence on Carrier System and Nucleotide Modification

“…Various non-viral gene delivery vehicles made of cationic polymers or lipids with different modifications have been developed 27,43 . However, beyond the transfection of established cell lines, only few of them achieved robust gene delivery in primary cells 22,44 . The number of successful gene transfer systems is even more limited, when delivery of specific cargo such as mRNA is demanded 45 .…”

Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery

“…Rationally designed targeting approaches, such as including ligands to tissue-specific receptors, are an alternative strategy. 94 Recently, Lou et al 95 reported an in vitro active targeting of sialic acid-ended glycoproteins on the surface of dendritic cells, mediated by a 30-amino-acid synthetic peptide with glutamic acid-alanine-leucine-alanine (GALA) repeats conjugated to polyplex carrying EGFP-mRNA. However, any opsonization on the LNPs forming a protein corona can be detrimental to active-targeting strategies, due to masking of the targeting ligands.…”

“…In this section, we discuss different delivery strategies and types of carriers used for mRNA-based protein therapy. 95,96 Intramyocardial injection of modified RNA encoding human VEGF-A, complexed with RNAiMAX, has been reported to improve heart function and long-term survival of mice with myocardial infarction. 101 Turnbull et al 102 compared intracoronary administration and direct myocardial injection of mRNA formulated into the LNPs (C14-113) in rodents and pigs, showing that the latter led to higher cardiac and lower off-target mRNA expression.…”

RGD-decorated cholesterol stabilized polyplexes for targeted siRNA delivery to glioblastoma cells

“…For pegylation, BCN-PEG (BCN-PEG-COOH) or BCN-PEG-cRGD as added to the polyplexes at the amount of PEG equivalent to 60 mol% of an azide of the polymer used for preparing polyplexes and left to react for 2 h at room temperature. This ratio was selected based on our prior studies, in which we investigated the optimal post-PEGylation ratio for RNA polyplexes [25,34]. For freezedrying of the polyplexes, sucrose was added to the polyplex formulation to a final concentration of 5% before snapfreezing in liquid nitrogen.…”

“…The resulting polymers formed small siRNA polyplexes through both polycation electrostatic interaction and siRNA and hydrophobic interaction via cholesterol. Subsequently, these polyplexes were post-modified with PEG-cRGD modified with bicyclo[6.1.0]nonyne (BCN) using copper-free click chemistry to shield surface charge and enable ligand-mediated cell binding and uptake [25,34]. In vitro experiments showed enhanced cellular uptake of PEG-cRGD modified polyplexes, which in turn resulted in improved transfection efficiency as compared to non-PEGylated particles.…”