The placenta is a multifaceted organ that plays a critical role in maintaining and protecting the developing fetus. Impaired implantation/placentation accounts for a number of obstetrical/neonatal complications, including recurrent miscarriage, placental accreta, preeclampsia, preterm delivery and intrauterine growth restriction, and it is our long-term goal to understand how individual genes and their products impacts normal and compromised placental function. Many of the questions remaining about the progression of human pregnancy cannot be answered directly, necessitating the use of appropriate animal models. While all animal models have merit, most suffer from the inability to repetitively sample blood from both the maternal and fetal side of the placenta, limiting their usefulness in the study of placental or fetal physiology under non-stressed in vivo conditions. The pregnant sheep has been used extensively to investigate maternal?fetal interactions due to the ability to surgically place catheters in both the maternal and fetal vasculature, allowing repeated sampling from non-anesthetized pregnancies. However, this animal model suffered from the inability to alter specific gene function in the placenta. Recently, we developed in vivo lentiviral-mediated RNA interference, by infecting the blastocyst cells that give rise to the placenta, and were able to limit the expression of ovine chorionic somatomammotropin (oCSH) during gestation. Placental and fetal weights were reduced 52% and 32%, respectively, near-term. In a second study, fetal weights were already reduced 20% at 50 days of gestation (dGA). While these data are exciting and support an important role for CSH in placental and fetal growth, there was variability in the resulting phenotypes, which likely reflects varying expression of the shRNA during the progression of gestation. The recent description of CRISPR (clustered regularly interspaced short palindromic repeat) sequences coupled with the single CRISPR associated endonuclease Cas9 (CRISPR- Cas9) to introduce double-strand breaks in target DNA of mammalian cells, typically followed by non- homologous end joining, could provide a new approach to permanently alter gene function in the placenta. By incorporating a ?guide RNA? in conjunction with a Cas9 expression cassette in a lentiviral vector, we propose that we can induce permanent gene mutations within the sheep placenta in vivo. Specifically, we will: 1) Test the in vitro efficiency of multiple lentiviral-oCSH CRISPR-Cas9 targeting vectors to functionally mutate both alleles of the oCSH gene, without off-target mutations.; and 2) Determine the efficiency of functionally ablating both alleles of the oCSH gene in vivo, through CRISPR-Cas9 targeting, as assessed at 35 days of gestation. This research is designed to test the hypothesis that: Genes expressed in the sheep placenta can be functionally mutated, thereby providing an animal model by which the function of specific genes can be studied in vivo in a non-stressed/non-anesthetized state. For this Small Grant (R03) we will: 1) develop new technology, and 2) test the efficiency of in vivo gene editing, which then can be applied to a number of species.
Functional placental insufficiency is a major cause of intrauterine growth restriction, leading to increased infant morbidity and mortality. We aim to develop direct gene editing within the sheep placenta, as a means to alter placental gene function, in an animal which lends itself to in depth in vivo studies of placental function and fetal development. However, this methodology should be applicable to a variety of species as well.
| Jeckel, K M; Boyarko, A C; Bouma, G J et al. (2018) Chorionic somatomammotropin impacts early fetal growth and placental gene expression. J Endocrinol 237:301-310 |
