Efficient Delivery of GSDMD‐N mRNA by Engineered Extracellular Vesicles Induces Pyroptosis for Enhanced Immunotherapy

Abstract

Pyroptosis is a newly discovered inflammatory form of programmed cell death, which promotes systemic immune response in cancer immunotherapy. GSDMD is one of the key molecules executing pyroptosis, while therapeutical delivery of GSDMD to tumor cells is of great challenge. In this study, an extracellular vesicles‐based GSDMD‐N mRNA delivery system (namely EVTx) is developed for enhanced cancer immunotherapy, with GSDMD‐N mRNA encapsulated inside, Ce6 (Chlorin e6 (Ce6), a hydrophilic sensitizer) incorporated into extracellular vesicular membrane, and HER2 antibody displayed onto the surface. Briefly, GSDMD‐N mRNA is translationally repressed in donor cells by optimized puromycin, ensuring the cell viability and facilitating the mRNA encapsulation into extracellular vesicles. When targeted and delivered into HER2+ breast cancer cells by the engineered extracellular vesicles, the translational repression is unleashed in the recipient cells as the puromycin is diluted and additionally inactivated by sonodynamic treatment as the extracellular vesicles are armed with Ce6, allowing GSDMD‐N translation and pyroptosis induction. In addition, sonodynamic treatment also induces cell death in the recipient cells. In the SKBR3‐ and HER2 transfected 4T1‐ inoculated breast tumor mouse models, the engineered EVTx efficiently induces a powerful tumor immune response and suppressed tumor growth, providing a nanoplatform for cancer immunotherapy.

Truncated PD1 Engineered Gas‐Producing Extracellular Vesicles for Ultrasound Imaging and Subsequent Degradation of PDL1 in Tumor Cells

“…[ 10 ] Shi et al developed a synthetic multivalent antibodies retargeted exosome (SMART‐Exo) displaying both anti‐human CD3 and anti‐human HER2 antibodies, and the SMART‐Exos dually targeting T cell CD3 and breast cancer‐associated HER2 receptors for immune‐activation. [ 11 ] EVs were also used to pack GSDMD‐N mRNA to induce cell pyroptosis in HER + breast cancer, [ 26 ] in turn the immunogenic pyroptosis induced a robust immunotherapy. Moreover, immune cell‐derived EVs and tumor cell‐derived EVs were used as antigen sources or adjuvant carriers to boost anti‐cancer immune response.…”

Recent advances in extracellular vesicles for therapeutic cargo delivery

“…Since puromycin inhibits translation, untranslated GSDMD-N mRNA can be easily sorted into exosomes. Through this process, they successfully loaded GSDMD-N mRNA into exosomes 68 . However, loading mRNA into nanocarriers is still challenging due to the instability and large size of the mRNA, which complicates packaging and controlled release.…”

Extracellular vesicles as carriers of mRNA: Opportunities and challenges in diagnosis and treatment

“…Although an effective technique to deliver GSDMD protein to cancer cells presents a challenge, EV-mediated delivery of functional GSDMD mRNA to cancer cells offers a promising solution. EVs expressing anti-HER2 + antibodies were used in a recent study to deliver GSDMD mRNA to HER2 + cancer cells in a HER2 overexpressing mouse model resulting in induced pyroptosis and a tumor immune response that significantly suppressed tumor growth and prolonged mouse survival compared to control mice that were treated with either PBS or unloaded EVs ( Figure 7 A-B ) 121 . This study demonstrated the feasibility of using EVs to deliver exogenous mRNA to tumors to induce tumor immune response to treat HER2 + breast cancer, setting the stage for engineered EVs to be used for other cancer immunotherapy targets.…”