(M1430-07-41) Endometrium-Targeted mRNA-LNPs for the Treatment of Infertility

Purpose: Dysfunction in the endometrium, the uterus lining where embryo attachment occurs, can cause infertility. Women with a thin endometrium (TE) are less likely to become pregnant. Regenerating the endometrium using cytokines is promising, but off-target toxicity and poor drug retention within the uterus are drawbacks. We hypothesized that messenger RNA (mRNA) delivery by lipid nanoparticles (LNPs) can induce and sustain protein delivery. However, an endometrium-targeted mRNA-LNP has not been described. The endometrium undergoes significant changes during the menstrual cycle [1]. During the window of implantation (WOI), integrin receptors are overexpressed to prepare the uterus for embryo implantation. Here, we leveraged the overexpressed integrins during the WOI to deliver mRNA to the endometrium by intrauterine infusion.

Methods: LNPs were formulated by rapid mixing. Treatment during the WOI was performed in mice on post-coitus day 5. Treatments were infused in the uterus following a laparotomy. Luciferase expression was quantified from tissue homogenates using a plate reader. GFP was observed using an epifluorescence microscope. Tissue levels of granulocyte macrophage-colony stimulating factor (GM-CSF) were measured by ELISA. TE mouse model was induced by intrauterine ethanol infusion.

Results: mRNA-LNPs were engineered to allow for high-density RGD ligand surface functionalization. Ligand-conjugated mRNA-LNP showed 4-fold higher luciferase expression in the uterus compared to untargeted LNP when infused during the WOI. There was no difference in uterus expression between ligand-conjugated and untargeted LNPs when the infusion was performed outside the WOI. Additionally, targeted LNPs showed very low luciferase expression in the liver or spleen, while the untargeted LNPs showed widespread expression in off-target organs, similar to previous reports [2, 3]. Microscopic analysis revealed that the GFP expression originated from endometrial epithelial and stromal cells.  A single intrauterine infusion of GM-CSF mRNA-loaded LNPs sustained protein production for 24 hr with minimal systemic exposure. GM-CSF mRNA enhanced embryo implantation rates in the TE model compared to untreated controls and recombinant protein-treated animals.

Conclusion: Our findings provide an effective and potentially safer method to deliver cytokines for treating TE. The LNP described here is the first endometrium-targeted mRNA delivery system and the first example of using mRNA for treating female infertility. The ligand-conjugated LNP can potentially deliver therapeutic nucleic acids in disease applications that show integrin overexpression, including cancers, inflammatory bowel disease, and endometriosis.

References: 1. Lessey, B. et al. J Clin Invest 90, 188-195 (1992).
2. Abbasi, S.; Masai, M. et al. Mol Ther 32, 1266-1283 (2024).
3. Van Guyse, J.; Abbasi, S. et al. Angew Chem Int Ed Engl, e202404972 (2024).

Acknowledgements: This work was supported by the National Institutes of Health (NIH) (grant no. R01HD103124, R01HD108905).