Intratumoral mRNA electrotransfer: establishment and preclinical proof of concept

BASIC DATA OF THE RESEARCH PROJECT

• ARRS code: J3-50110
• TITLE: Intratumoral mRNA electrotransfer: establishment and preclinical proof of concept
• PRICE CLASS: C
• PROJECT LEADER: Katja Uršič Valentinuzzi, PhD
• APPLICANT RESEARCH ORGANISATION: Institute of Oncology Ljubljana
• COLLABORATOR: University Clinic Golnik
• DURATION: 1. 10. 2023 – 30. 9. 2026
• FINANCING: Slovenian Research and Innovation Agency

Problem identification

mRNA therapeutics comprise a rapidly expanding category of drugs that will change the standard of care for many diseases including cancer. Currently, ex vivo mRNA delivery in cells has reached Phase III clinical testing. On the contrary, in vivo delivery, especially intratumoral delivery, is lagging behind. Therefore, we found there is a lot of room for improvement and believe that electroporation could be exploited as a delivery method for intratumoral mRNA immunotherapy. To highlight, naked mRNA molecules will be electroporated, avoiding the never-ending optimization of carrier vehicles as liposomes. Moreover, this is the first time, to our knowledge, that the intratumoral electrotransfer of trans-amplifying mRNAs will be investigated in vivo. Collectively, intratumoral mRNA electrotransfer would increase in situ bioavailability of therapeutics and at the same time minimize systemic side effects. In the project, we will use reporter mRNA molecules to optimize electric pulse parameters and investigate pharmacokinetics, toxicity, tolerance as well as immunogenicity of the intratumoral mRNA electrotransfer. The effectiveness of mRNA electrotransfer will be compared to plasmid electrotransfer. Additionally, warming-up immunologically cold B16F10 melanoma by intratumoral electrotransfer of two therapeutic mRNAs would serve as a preclinical proof concept and will pave the way for the translation of intratumoral mRNA electrotransfer in clinical testing.

Objectives

The mission is to challenge intratumoral mRNA electrotransfer. The three objectives are (see the picture below).

We intend to develop strategies for intratumoral mRNA electrotransfer by optimizing electroporation with specific emphasis on electric pulse parameters. Therefore, both, the reporter and the therapeutic state-of-the art mRNA molecules will be designed and investigated in in vitro and in vivo setting. For pharmacological and toxicology studies, state-of-the-art in vivo imaging systems and droplet digital PCR will be used. Finally, as a proof of concept, the idea of dual intratumoral mRNA electrotransfer will be investigated in murine B16F10 malignant melanoma. We intend to upgrade in vivo mRNA electroporation approach by two additional details; controlled electroporation-induced immune response and mRNA encoding nanobodies instead of antibodies. The project will pave the way for the use of intratumoral mRNA electrotransfer in cancer treatment in both, our laboratory and worldwide.

The aims of the project will be implemented through Work Packages (WP) divided into Specific Tasks (ST).

WP 1: mRNA molecules (1-6 month)
ST 1.1: Custom mRNA molecules design (1-6 month)

WP 2: mRNA electrotransfer (6-24 month)
ST 2.1: In vitro mRNA electrotransfer (6-18 month)
ST 2.2: In vivo mRNA electrotransfer (month 12-24)
ST 2.3: Pharmacokinetics, toxicity, tolerance and immunogenicity (month 12-24)

WP 3: Preclinical proof of concept: Dual intratumoral mRNA electrotransfer (18-30 month)
ST 3.1: Dual intratumoral mRNA electrotransfer and its local effects (month 18-30)
ST 3.2: Dual intratumoral mRNA electrotransfer and its protective potential (month 18-30)

WP4: Project management (0-36 month)
ST 4.1 Documentation preparation, permissions
ST 4.2 Project progress monitoring, evaluation and interpretation of results
ST 4.3 Dissemination and communication
ST 4.4 Preparation of scientific and financial reports

Main deliverables:

• Five custom-designed mRNA molecules including reporter mRNAs therapeutic mRNAs as well as replicase-encoding mRNA enabling trans-replication of any listed mRNA molecule in tumors.
• Optimized intratumoral mRNA electrotransfer with investigated pharmacokinetics, toxicity, tolerance and immunogenicity.
• Dual mRNA electrotransfer for treatment of melanoma.

Main impact

The project will bridge the gap between two technologies; mRNA and in vivo electroporation of tumors. The intratumoral mRNA electrotransfer will thus expand the armamentarium of mRNA-based therapeutic approaches in oncology.