Exploring Cytotoxic mRNAs as a Novel Class of Anti-cancer Biotherapeutics

Abstract

New treatments to overcome the obstacles of conventional anti-cancer therapy are a permanent subject of investigation. One promising approach is the application of toxins linked to cell-specific ligands, so-called immunotoxins. Another attractive option is the employment of toxin-encoding plasmids. However, immunotoxins cause hepatoxicity, and DNA therapeutics, among other disadvantages, bear the risk of insertional mutagenesis. As an alternative, this study examined chemically modified mRNAs coding for diphtheria toxin, subtilase cytotoxin, and abrin-a for their ability to reduce cancer cell growth both in vitro and in vivo. The plant toxin abrin-a was the most promising candidate among the three tested toxins and was further investigated. Its expression was demonstrated by western blot. Experiments with firefly luciferase in reticulocyte lysates and co-transfection experiments with EGFP demonstrated the capability of abrin-a to inhibit protein synthesis. Its cytotoxic effect was quantified employing viability assays and propidium iodide staining. By studying caspase-3/7 activation, Annexin V-binding, and chromatin condensation with Hoechst33258 staining, apoptotic cell death could be confirmed. In mice, repeated intratumoral injections of complexed abrin-a mRNA resulted in a significant reduction (89%) of KB tumor size compared to a non-translatable control mRNA.

mRNA as novel technology for passive immunotherapy

“…Most if not all groups purified their IVT mRNA before in vivo administration. While some simply precipitated the mRNA [195,227,236], most used commercial purification kits. Only a few researchers applied HPLC purification [188,218,232].…”

“…Thereafter, it took almost two decades until further studies started to demonstrate the broad potential of mRNA-based protein therapies. Meanwhile, there is a plethora of publications on a huge variety of indications comprising anemia [ 188 , 218 ], hemophilia [ 223 , 224 ], myocardial infarction [ 155 , 225 ], cancer [ 226 , 227 ], lung disease such as surfactant B deficiency and asthma [ 228 – 230 ], metabolic disorders [ 231 – 235 ], fibrosis [ 195 ], skeletal degeneration [ 236 ], tendon impairment [ 237 ], and neurological disorders such as sensory nerve dysfunction, Friedreich’s ataxia and Alzheimer’s disease [ 238 – 240 ]. Whereas evidence for the therapeutic potential of mRNA is mostly restricted to mouse models, first data in swine indicate that mRNA-based protein therapies are feasible also in large animals [ 218 , 225 ].…”

“…Regarding the cap structure, almost all early studies cotranscriptionally generated cap0 mRNAs using ARCA [ 226 , 228 , 229 ]. However, since about 2 years, research groups prefer to apply mRNAs with a cap1 5′-end [ 223 , 227 , 231 ].…”

Nano‐Oncologicals: A Tortoise Trail Reaching New Avenues

“…[199,200] As mRNAs act transiently in the cytoplasm and do not have the danger of genome integration, they have gained momentum as a safe alternative to conventional gene therapy. In cancer, mRNAs encoding antibodies, [200][201][202][203] tumor suppressor proteins, [204,205] toxins, [206] suicide genes, [207] Table 6. Relevant anticancer oligonucleotide-based nanosystems currently in clinical trials.…”

“…Similarly, intratumoral administration of mRNA encoding a toxin (A-chain of abrin-a toxin) resulted in a significantly reduced tumor growth in a papilloma mouse model. [206] More examples and clinical trials using anticancer mRNA are discussed in later sections.…”