Poster W13 - In Utero Gene Delivery in Fetal Rhesus Monkeys Mediated by Polymeric Nanoparticles

Introduction: In utero gene therapy has the potential to correct genetic diseases during early stages of development and prior to the onset of pathology. Non-viral lipid- and polymer-based vehicles have shown promise for nucleic acid delivery in vitro and in vivo. However, the primary barrier for clinical translation remains delivery to target tissues in vivo. Poly(lactic-co-glycolic acid) (PLGA), for example, is widely used as a drug carrier. This material is biodegradable and non-toxic with demonstrated use for in utero delivery (1). However, PLGA nanoparticles (NPs) are inefficient at nucleic acid delivery compared to other NP systems. Poly(amine-co-esters) (PACEs), on the other hand, are mildly cationic and have been shown to have a high encapsulation efficiency of nucleic acids, as well as a high cellular association, transfection, and tolerability in vitro and in vivo, supported by studies in small animal models (2,3). However, for future clinical translation, it is essential that these NPs are validated for safety and efficiency in a primate model that is developmentally and physiologically similar to humans. This study demonstrates safe and successful NP administration and gene delivery to multiple organs in fetal rhesus monkeys during early gestation.

Materials and

Methods: A PACE-polyethylene glycol (PEG) block co-polymer was synthesized using enzyme-catalyzed copolymerization, as described previously (2). Esther-terminated 50:50 PLGA with inherent viscosity 0.55–0.75 (dL/g) was obtained commercially. PLGA and PACE-PEG NPs encapsulating fluorescent DiD dye were formulated by single emulsion solvent evaporation (2). PACE-PEG NPs encapsulating mRNA for EGFP were formulated by double emulsion solvent evaporation. PLGA DiD NPs, PACE-PEG DiD NPs, and PACE-PEG EGFP mRNA NPs prepared under sterile conditions and verified endotoxin free were administered to fetal rhesus monkeys in the early second trimester via the portal vein (4). Biodistribution of DiD-loaded NPs was assessed 24 hours after administration and EGFP expression was assessed in mRNA-loaded NPs 48 hours after administration. Tissues collected included the following: brain, thymus, heart, lung, omentum, spleen, pancreas, liver, adrenals, kidneys, gonads, skin, muscle, bone, gastrointestinal (GI) tract, and placenta (5). Whole organ fluorescence imaging for biodistribution studies in multiple tissues was performed using an In Vivo Imaging System (IVIS). Tissues were flash-frozen and frozen sections were analyzed by quantitative microscopy using an EVOS Cell Imaging System and a custom MATLAB image analysis program.

Results, Conclusions, and Discussions: PLGA DiD NPs exhibited accumulation primarily in the fetal liver and spleen, similar to mouse studies. However, PACE-PEG DiD NPs exhibited more widespread biodistribution (Figure 1). In addition to the liver and spleen, PACE-PEG NPs were also found in the thymus, lung lobes, GI tract, adrenal glands, and kidneys. The placenta did not indicate any NP uptake and observations for all fetuses were within normal limits with no evidence of adverse findings. We also studied the relationship between delivery and transfection efficiency of nucleic acid cargo by administering PACE-PEG NPs encapsulating EGFP mRNA. Several tissues demonstrated detectable EGFP expression, which was assessed as a measurable green fluorescence signal in tissues from animals treated with PACE-PEG EGFP mRNA NPs.

These studies demonstrate that nucleic acid-based therapeutics can be effectively delivered in utero using PACE polymeric vehicles that are designed for gene delivery. Compared to PLGA NPs, PACE-PEG NPs offer high nucleic acid encapsulation efficiency and a wider biodistribution profile. By reaching multiple fetal tissues, including the lungs, pancreas, and GI tract, PACE-PEG NPs provide a potential therapeutic pathway for genetic diseases such as cystic fibrosis. Findings support polymeric NPs as candidates for safe, effective fetal delivery of therapeutic nucleic acids and provides evidence for feasible clinical translation of this innovative technology for the treatment of genetic disorders.

Acknowledgements (Optional):

References:
(1) Ricciardi A, et al., Nature Communications. 2018.
(2) Kauffman A and Piotrowski-Daspit AS, et al. Biomacromolecules. 2018.
(3) Grun M, et al. Biomaterials. 2021.
(4) Tarantal AF. In: The Laboratory Primate, Chapter 20. 2005.
(5) Jimenez DF, et al., Gene Therapy. 2005.