Over the past two decades, chemists have successfully developed pseudouridine and its derivatives to synthesize messenger RNA (mRNA) with lower immunogenicity and better translational efficiency. This breakthrough has fueled global interest in mRNA-based therapeutics, as mRNA allows for the temporary expression of immune-activating proteins without altering a person’s genetic makeup. Another key advancement has been the development of lipid nanoparticles (LNPs), which enable the efficient delivery of mRNA into target cells. This scalable technology played a crucial role in the rapid development of the COVID-19 vaccines. These mRNA-based vaccines, which use LNPs for delivery, were the first to be approved during the pandemic and demonstrated over 90% effectiveness. Following this success, researchers explored numerous mRNA-based vaccines for other diseases, including cancer. While cancer vaccines offer several advantages over other immunotherapies, their effectiveness has been limited, often benefiting only a subset of patients. Nonetheless, continued advancements in mRNA-based cancer vaccines hold great promise for improving cancer treatment in the future. Cancer vaccines have the potential to activate a new wave of tumor-killing T cells, providing an advantage over checkpoint blockade therapy, which is effective primarily for inflamed cancers already infiltrated by activated T cells. Additionally, cancer vaccines can target a broader range of intracellular cancer-specific proteins called ‘antigens’, addressing a major limitation of CAR T cell therapy, which is only effective against cancers with specific surface antigens. These vaccines work by delivering mRNA encoding antigenic proteins into the cytoplasm of immune cells, mainly macrophages and dendritic cells, where the mRNA is translated into antigen, which further activates cancer-killing T cells and B cells. The success of a therapeutic cancer vaccine depends on the selection of appropriate antigens against specific cancers. However, in the case of cancer, these tumor-associated antigens delivered via traditional delivery vehicles may not be seen as a threat by the immune system that can recruit immune cells, because they do not provide strong danger signals. Therefore, adjuvants are incorporated into the vaccines. In this work, we hypothesized that guanine- and cytosine-rich double-stranded DNA (GC-rich dsDNA) could serve as an endogenous danger signal, acting as an adjuvant in cancer vaccines. Previously used adjuvants were known to recruit immune cells near the targeted cells through various metabolic pathways, culminating in the release of cell-signaling chemokines and cytokines. One such widely studied metabolic pathway is the NF-κB pathway that promotes the formation of inactive pro-IL-1β. In presence of a second stimuli such as lysosomal rupture or Potassium Efflux or Calcium Influx or Mitochondrial Reactive Oxygen Species, some inactive proteins oligomerize to form a protein complex called the inflammasome, namely NOD-like receptor family pyrin domain-containing 3 (NLRP3) Inflammasome inside the cytoplasm of an immune cell such as a macrophage. Inflammasome activation leads to the cleavage of inactive pro-IL-1β into its active form, IL-1β, and promotes pore formation on the surface of macrophages. This process facilitates the secretion of cytokines—particularly IL-1β—that attract immune cells, thereby enhancing immunogenicity. dsDNA is known to activate the Absent in Melanoma 2 (AIM2) inflammasome. However, dsDNA can have an off-target effect by activating the Stimulator of Interferon Genes (STING) pathway, resulting in the secretion of Type I Interferons (IFNs). These Type I IFNs are known to attenuate mRNA translation. To address this issue, we performed a monoclonal antibody-based combination approach, in which we blocked Type I IFN-sensing IFNAR receptors on macrophages and then administered the nanoparticles co-encapsulating dsDNA and mRNA. The nanoparticle formulation consisted of gold-standard lipids, including an ionizable lipid (SM-102), a helper lipid (DSPC), a stabilizing lipid (cholesterol), and a PEGylated lipid (DMG-PEG-Amine), in a ratio of 50:10:38.5:1.5, respectively. The nanoparticles had an average size of approximately 180 nm and exhibited a nearly neutral zeta potential. They remained stable for up to one week. Encapsulation efficiencies for mRNA and GC-rich dsDNA were 75% and 65%, respectively, as confirmed by Ribogreen and Picogreen assays. As a proof of concept, we used commercially available green fluorescent protein (GFP)-encoding mRNA and GC-rich dsDNA, Poly(dG:dC), both encapsulated within the lipid nanoparticles and delivered along with a commercially available antibody, anti-mouse IFNAR-1 (Clone: MAR1-5A3). Remarkably, we observed a threefold increase in mRNA translation after treating with the IFNAR-blocking monoclonal antibody, as confirmed by flow cytometry. Additionally, there was an 11.5-fold increase in IL-1β-mediated immunogenicity, as measured by IL-1β ELISA in lipopolysaccharide (LPS)-primed immortalized bone marrow-derived macrophages (iBMDMs) from mice. IL-1β cytokine levels due to GC-rich dsDNA were compared with those induced by a well-known danger signal, adenosine triphosphate (ATP). To validate the involvement of AIM2 inflammasome activation by dsDNA, experiments were also conducted in NLRP3 and caspase-knockout iBMDMs. The efficacy of the cancer vaccines was enhanced by increasing adjuvanticity without compromising mRNA translation. To our knowledge, this is the first study to explore the use of GC-rich dsDNA and an IFNAR-blocking antibody to improve cancer vaccine efficacy in this manner.
