Extracellular Vesicle–Mediated In Vitro Transcribed mRNA Delivery for Treatment of HER2+ Breast Cancer Xenografts in Mice by Prodrug CB1954 without General Toxicity

Abstract

Prodrugs are harmless until activated by a bacterial or viral gene product; they constitute the basis of gene-delivered prodrug therapies called GDEPT, which can kill tumors without major side effects. Previously, we utilized the prodrug CNOB (C 16 H 7 CIN 2 O 4 ; not clinically tested) and enzyme HChrR6 in GDEPT to generate the drug MCHB (C 16 H 9 CIN 2 O 2) in tumors. Extracellular vesicles (EVs) were used for directed gene delivery and HChrR6 mRNA as gene. Here, the clinical transfer of this approach is enhanced by: (i) use of CB1954 (tretazicar) for which safe human dose is established; HChrR6 can activate this prodrug. (ii) EVs delivered in vitro transcribed (IVT) HChrR6 mRNA, eliminating the potentially harmful plasmid transfection of EV producer cells we utilized previously; this has not been done before. IVT mRNA loading of EVs required several steps. Naked mRNA being unstable, we ensured its prodrug activating functionality at each step. This was not possible using tretazicar itself; we relied instead on HChrR6's ability to convert CNOB into MCHB, whose fluorescence is easily visualizable. HChrR6 mRNA-translated product's ability to generate fluorescence from CNOB vicariously indicated its competence for tretazicar activation. (iii) Systemic IVT mRNA-loaded EVs displaying an anti-HER2 single-chain variable fragment ("IVT EXO-DEPTs") and tretazicar caused growth arrest of human HER2 þ breast cancer xenografts in athymic mice. As this occurred without injury to other tissues, absence of off-target mRNA delivery is strongly indicated. Many cancer sites are not amenable for direct gene injection, but current GDEPTs require this. In circumventing this need, a major advance in GDEPT applicability has been accomplished.

Nanoplatforms for mRNA Therapeutics

“…Tretazicar (CB1954, a prodrug in clinical trial) was used in another EV‐based system. [ 161 ] Different from the abovementioned work where mRNA loaded EVs were harvested from 293FT cells transfected with pXPort/HChrR6 plasmid, [ 159 ] mRNA loaded‐EVs were produced from PEI‐mRNA polyplex treated HEK293 cells directly in the tretazicar/EXO‐DEPT system.…”

“…Advances in mRNA delivery platforms make new approaches in cancer treatment possible. There are multiple forms of mRNA-based cancer therapy including suicide gene/prodrug system, [147,161] reprogramming tumor suppressor gene, [216] and introduction of tumor suppressors. [145,146,217] Restoring tumor suppressor genes can not only induce tumor cell apoptosis, but also sensitize tumor cells to chemotherapy.…”

Extracellular vesicles and oncogenic signaling

“…Gomari et al [168] additionally reported a significant reduction in tumor growth by the administration of targeted Exos but not of free or untargetedexosomal doxorubicin in a murine breast cancer model. Wang and Forterre and colleagues investigated in the context of HER2 + breast cancer feasible and safe administration options to specifically guide prodrug/enzyme regimens to cancer cells with minimal off-target toxicity [169,170]. They also used EVs for specific targeting of only the HER2 + subpopulation through a chimeric protein designed to be presented at the EV surface.…”

“…They also used EVs for specific targeting of only the HER2 + subpopulation through a chimeric protein designed to be presented at the EV surface. Therefore, they successfully administered in vitro-transcribed mRNA through EVs [169,170].…”

Perspectives in Manipulating EVs for Therapeutic Applications: Focus on Cancer Treatment

“…As a result, EVs were able to deliver mRNA to the cells of HER2-positive human breast tumor xenografts in a targeted manner due to the surface-expressed anti-HER2 antibody, which inhibited the growth of the xenografts in mice [ 50 ]. Interestingly, the later results suggested that EVs may deliver in vitro transcribed enzyme-encoding mRNA, which allows to eliminate the potentially harmful plasmid transfection of EV-parental cells [ 51 ]. Another interesting possibility was proposed by Sancho-Albero et al [ 31 ].…”