Engineering therapeutic messenger RNA for triple-negative breast cancer

Background: Targeted therapies are revolutionising the landscape of cancer treatment, providing better therapeutic responses and minimising the adverse effects of conventional treatments such as radiation and chemotherapy. Human epidermal growth factor receptor 2 (HER2) is overexpressed in multiple cancer types and remains one of the therapeutic flagship targets in oncology. The HER2-targeting monoclonal antibody trastuzumab has dramatically improved the survival of patients with HER2-positive breast cancer. More recently developed antibody-drug conjugates, such as trastuzumab deruxtecan (T-DXd), have demonstrated unprecedented efficacy in HER2-overexpressing cancers. However, only a small subset of patients can benefit from HER2-targeted treatments (15-30% of breast cancers). Triple-negative breast cancer (TNBC) is characterised by the absence of HER2 and hormone receptors. TNBC is an aggressive subset of breast cancers for which few targeted therapies are available. It has recently been shown that artificial overexpression of HER2 could sensitise TNBC xenograft models to trastuzumab, however the clinical relevance of this strategy was limited by the use of viral vectors. Messenger RNA (mRNA) technology has been cemented as a safe and effective therapeutic modality, as evidenced by its success in SARS CoV-2 vaccination. Its potential to revolutionise cancer therapy is now becoming increasingly evident. We hypothesised that mRNA technology could be used to transiently overexpress HER2 in TNBCs to sensitise them to anti-HER2 therapeutics. Methods: Molecular cloning techniques were used to engineer a plasmid vector for in vitro transcription (IVT) of a truncated variant of HER2 (TrHER2), lacking the intracellular domain (ICD) to eliminate the potential for proliferative signalling. Modified nucleosides were incorporated into IVT reactions and mRNA was enzymatically capped and poly(A) tailed. TrHER2 and control (Luciferase and WtHER2) mRNA were delivered to TNBC cells using conventional transfection reagents. TrHER2 protein expression and HER2 signalling were measured by means of western immunoblot. TrHER2 localisation was assessed by immunofluorescent microscopy and flow cytometry. The impact of TrHER2 expression on migration and clonogenicity was measured by scratch and colony formation assays. Trypan blue and CellTiter-Fluor assays were used to assess the impact of TrHER2 expression on the efficacy of T-DXd and trastuzumab. Induction of apoptosis was determined by flow cytometry with Annexin V and propidium iodide stain. The effect of TrHER2 mRNA on trastuzumab function was determined by co-culture of TNBC cells with human immune cells. Results: TrHER2 plasmids were engineered and validated by sanger sequencing. IVT reactions with these plasmids produced pure, high yield TrHER2 mRNA. TrHER2 protein was shown to be approximately 100 kDa, lacking expression of the HER2 ICD. TrHER2 mRNA expression demonstrated cell surface localisation with a >110-fold increase in expression, exceeding that of WtHER2 mRNA (50-fold) at an equal dose. TrHER2 mRNA delivery did not active the HER2 signalling pathway, instead reducing cell migration and clonogenicity compared to WtHER2 mRNA. TrHER2 mRNA expression significantly increased the efficacy of T-DXd, causing an 80% reduction in viability (p<0.0001) by inducing DNA damage and apoptosis. TrHER2 mRNA also sensitised TNBC cells to antibody-dependent cell-based cytotoxicity by trastuzumab, causing an 37% reduction in viability compared to cells transfected with reporter mRNA (p<0.001). Significance: These findings demonstrate proof-of-concept for utilising mRNA-based overexpression as a strategy to enhance the sensitivity of TNBC cells to anti-HER2 therapies. Additionally, the results validate the safety profile of the engineered TrHER2 mRNA. The observed high efficacy suggests promising in vivo effectiveness. When paired with a suitable delivery system, this approach could have the potential for clinical translation.

Grantor dc:publisher

University College Cork

Year dc:date.issued

2024

Author dc:creator

• Leahy, Mark

Advisors dc:contributor.advisor

• Krajewska, Malgorzata
• Kowalski, Piotr
• O'Connor, Rosemary

• Messenger RNA
• Nanotechnology
• mRNA delivery
• Combination therapy
• Triple-negative breast cancer

Statement dc:rights

• © 2024, Mark Leahy.

Licence dc:rights.uri

• https://creativecommons.org/licenses/by-nc-nd/4.0/

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10468/17028

OAI identifier oai:identifier

oai:cora.ucc.ie:10468/17028