Invited talk 2
Dr. Lior ZANGI

Lior Zangi, PhD, is an Associate Professor with Tenure at the Icahn School of Medicine at Mount Sinai, New York. He completed his education and training at the Weizmann Institute of Science, and Harvard University. Studies methods for delivering modified mRNA (modRNA) to the heart to induce cardiac regeneration, protection, or cardiovascular regeneration post ischemic injury.  He has established a new method, modRNA based, for minimal invasive gene delivery into specific cell types and organs. In the last few years, these modRNA technology have been used for COVID19 vaccinations and promoting cardiovascular regeneration in ischemic heart disease. Currently, Prof. Zangi’s laboratory investigates mRNA delivery method into healthy or unhealthy specific cell types and organs, to fight different diseases such as heart failure and cancer.

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Topic:  The SMRTs* way for treating breast cancer.

*SMRTs - Specific Modified mRNA Translation System​

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Background: Triple negative breast cancer (TNBC) presents the worst prognosis among breast cancers with 40% mortality rate within 5 years from diagnosis. Due to the molecular characteristics of TNBC, cytotoxic chemotherapy remains the main form of treatment. An efficient targeted therapeutic approach is needed to improve treatment outcome with reduced toxicity. Here we developed a novel system that enable us to upregulate specific gene in TNBC mouse model (4T1 mouse model) to show the therapeutic value of mTOR inhibitor gene,  Pip4k2c together with a-CTLA-4 antibodies that was delivered using modified mRNA (modRNA) technology.

 

Material and methods: Breast cancer specific modified RNA translational system (Tumor SMRTs) is based on simultaneous delivery of two modRNAs: a suppressor gene (Cas6) carrying brest tumor-specific microRNA and a gene of interest carrying a Cas6 recognition site (hairpin). Upon Cas6 modRNA translation, the Cas6 protein attaches and cuts the hairpin present on the 5’UTR of the modRNA gene of interest. Only breast tumor cells express specific miRs, which detect the recognition element on the 3’UTR of the Cas6 modRNA and promote its degradation. This Cas6 modRNA degradation in breast tumor cells leads to successful translation of the gene of interest modRNA. For systemic delivery, therapeutic or reporter modRNA was encapsulated in lipid nanoparticles (LNP) and visualized using a bioluminescence (BL) imagining system. To assess tumor growth inhibition, Pip4k2c Tumor SMRTs alone or combined with anti-CTLA-4 modRNA (modRNab) was intravenously injected into female BALBc mice bearing 4T1 tumors every 3 days with 5 total injections.

 

Results: Four out of five of the designed nGFP breast cancer SMRTs  showed expression in 4T1 cells and significant decrease in mouse mammary gland epithelial cell line. Next, constructs carrying Luciferase (Luc) gene were directly injected into 4T1 mammary gland tumors and the contralateral femur skeletal muscle of BALBc mice. While Luc modRNA expressed in both tumor and femur skeletal muscle, the Luc breast cancer SMRTs expressed exclusively in the tumor. For systematically delivered modRNA encapsulate in NLP shown expression in the tumor and all major organs, however, Luc Tumor SMRTs shown significantly decreased expression in the liver (p<0.0001), spleen (p= 0.0009) and kidney (p= 0.0306). a-CTLA-4 antibody was produced using modRNA and detected in cell supernatants from HEK293T, 4T1 and epithelial cells 24h after transfection with modRNab. Serum a-CTLA-4 ab levels of mice injected with modRNab_LNPs were detected at least 11 days post injection. Importantly, delivery of Pip4k2c tumor SMRTs and modRNab encapsulated in NLP resulted in significant tumor growth inhibition when compared to non-treated mice (p= 0.0002) or Luc Tumor SMRTs injected mice (p= 0.0032).

 

Conclusions:

Tumor SMRTs allow specific gene expression in breast cancer tumor cells in vitro and in vivo. modRNA can be used for efficient production of monoclonal antibodies. LNP encapsulation allows efficient for systemic delivery of modRNA. Notably, delivery of Pip4k2c tumor SMRTs and modRNab encapsulated in NLP resulted in significant inhibition of breast tumor growth. We are confident that our SMRTs non-viral constructs could be created to achieve similar delivery to other tumor cell types, and that such methods will eventually preclude the reliance on viral vectors for cancer therapeutics.

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