Development of Non-Viral, Trophoblast-Specific Gene Delivery for Placental Therapy

Abstract

Low birth weight is associated with both short term problems and the fetal programming of adult onset diseases, including an increased risk of obesity, diabetes and cardiovascular disease. Placental insufficiency leading to intrauterine growth restriction (IUGR) contributes to the prevalence of diseases with developmental origins. Currently there are no therapies for IUGR or placental insufficiency. To address this and move towards development of an in utero therapy, we employ a nanostructure delivery system complexed with the IGF-1 gene to treat the placenta. IGF-1 is a growth factor critical to achieving appropriate placental and fetal growth. Delivery of genes to a model of human trophoblast and mouse placenta was achieved using a diblock copolymer (pHPMA-b-pDMAEMA) complexed to hIGF-1 plasmid DNA under the control of trophoblast-specific promoters (Cyp19a or PLAC1). Transfection efficiency of pEGFP-C1-containing nanocarriers in BeWo cells and non-trophoblast cells was visually assessed via fluorescence microscopy. In vivo transfection and functionality was assessed by direct placental-injection into a mouse model of IUGR. Complexes formed using pHPMA-b-pDMAEMA and CYP19a-923 or PLAC1-modified plasmids induce trophoblast-selective transgene expression in vitro, and placental injection of PLAC1-hIGF-1 produces measurable RNA expression and alleviates IUGR in our mouse model, consequently representing innovative building blocks towards human placental gene therapies.

Fig 1. (A) The HPMA-DMAEMA copolymer used for DNA delivery in both in vitro and in vivo studies. (B) Maps of the CMV-eGFP and Trophoblast-specific plasmids.

Fig 2. Representative images of (A) GFP expression following transfection of BeWo with plasmid alone or (B) CMV-eGFP nanoparticles demonstrate greater transgene expression following complex formation than plasmid alone. (C) GFP expression in BeWo cells after transfection with nanoparticles containing PLAC1-GFP or (D) CyP19a-923-GFP for 4 days, resulted in comparable transgene expression to the global CMV promoter, similar results were seen on 4 different passages. Cell nuclei are stained with Dapi (blue). Proliferation levels (E) and apoptosis levels (F) in BeWo cells were not changed when cells were incubated with nanocomplexes for 48 hours compared to BeWo cells incubated with eGFP plasmid alone for the same time period, mean +/- SEM, n>4 passages per treatment.

Fig 3. GFP expression in Human uterine fibroblasts after transfection with nanoparticles containing (A) CMV-GFP, (B) PLAC1-GFP or (C) CyP19a-923-GFP. HEK293 cells transfected with (D) CMV-GFP, (E) PLAC1-GFP or (F) CyP19a-923-GFP demonstrated low but visible transgene expression under the CMV promoter but no GFP expression with the trophoblast-specific promoters. GFP expression in HPMVECs cells after transfection with nanoparticles containing (G) CMV-GFP, (H) PLAC1-GFP or (I) CyP19a-923-GFP. Representative images seen on 4 different passages for each cell type.

Cell nuclei are stained with Dapi (blue).

Fig 4. Offspring birthweights at delivery were the same in Sham-operated, Internal control and UABL + PLAC1-HuIGF-1 nanoparticle treated group, however the Uterine Artery Branch Ligation, and Cyp19a-HuIGF-1 nanoparticle treated groups had significantly lower birthweights.

N>6 dams per group, ANOVA<0.001, post-hoc Tukeys *<0.05, **<0.01.

Fig 5. Placental morphology in the Labyrinthine (L) zone in (A) Sham, (B) UABL,(C) NP-PLAC1-hIGF-1treated groups is similar whereas differences in morphology can be seen in the NP-Cyp19a-hIGF-1 (D) treated group. (Magnification 40X). (E) The ratio of placental Junctional zone (Jz) area to Labyrinthine zone (Lz) area demonstrates significant expansion of the junctional zone in placentas treated with NP-Cyp19a-hIGF-1.