Nanoparticle-mediated delivery of placental gene therapy via uterine artery catheterization in a pregnant rhesus macaque.

Nanoparticles offer promise as a mechanism to non-invasively deliver targeted placental therapeutics. Our previous studies utilizing intraplacental administration demonstrate efficient nanoparticle uptake into placental trophoblast cells and overexpression of human IGF1 ( hIGF1 ). Nanoparticle-mediated placental overexpression of hIGF1 in small animal models of placental insufficiency and fetal growth restriction improved nutrient transport and restored fetal growth. The objective of this pilot study was to extend these studies to the pregnant nonhuman primate and develop a method for local delivery of nanoparticles to the placenta via maternal blood flow from the uterine artery. Nanoparticles containing hIGF1 plasmid driven by the placenta-specific PLAC1 promoter were delivered to a mid-gestation pregnant rhesus macaque via a catheterization approach that is clinically used for uterine artery embolization. Maternal-fetal interface, fetal and maternal tissues were collected four days post-treatment to evaluate the efficacy of hIGF1 treatment in the placenta. The uterine artery catheterization procedure and nanoparticle treatment was well tolerated by the dam and fetus through the four-day study period following catheterization. Nanoparticles were taken up by the placenta from maternal blood as plasmid-specific hIGF1 expression was detected in multiple regions of the placenta via in situ hybridization and qPCR. The uterine artery catheterization approach enabled successful delivery of nanoparticles to maternal circulation in close proximity to the placenta with no concerns to maternal or fetal health in this short-term feasibility study. In the future, this delivery approach can be used for preclinical evaluation of the long-term safety and efficacy of nanoparticle-mediated placental therapies in a rhesus macaque model. Highlights: Novel method to deliver therapeutics to maternal-fetal interfaceDelivery of nanoparticles to the placenta via maternal catheterization.

Placental Nanoparticle-mediated IGF1 Gene Therapy Corrects Fetal Growth Restriction in a Guinea Pig Model.

Fetal growth restriction (FGR) caused by placental insufficiency is a major contributor to neonatal morbidity and mortality. There is currently no in utero treatment for placental insufficiency or FGR. The placenta serves as the vital communication, supply, exchange, and defense organ for the developing fetus and offers an excellent opportunity for therapeutic interventions. Here we show efficacy of repeated treatments of trophoblast-specific human insulin-like 1 growth factor ( IGF1 ) gene therapy delivered in a non-viral, polymer nanoparticle to the placenta for the treatment of FGR. Using the guinea pig maternal nutrient restriction model of FGR, nanoparticle-mediated IGF1 treatment was delivered to the placenta via ultrasound guidance across the second half of pregnancy, after establishment of FGR. This treatment resulted in correction of fetal weight in MNR animals compared to control, improved fetal physiology and no negative maternal side-effects. Overall, we show for the first time a therapy capable of improving the entire pregnancy environment: maternal, placental, and fetal. This combined with our previous studies using this therapy at both mid pregnancy and in numerous cell and animal models demonstrate the plausibility of this therapy for future human translation to improve health outcomes of neonates and decrease numerous morbidities associated with the developmental origins of disease.

Maternal, placental and fetal response to a non-viral, polymeric nanoparticle gene therapy in nonhuman primates.

Background: Currently, there are no placenta-targeted treatments to alter the in utero environment. Water-soluble polymers have a distinguished record of clinical relevance outside of pregnancy. We have demonstrated the effective delivery of polymer-based nanoparticles containing a non-viral human insulin-like 1 growth factor ( IGF1 ) transgene to correct placental insufficiency in small animal models of fetal growth restriction (FGR). Our goal was to extend these studies to the pregnant nonhuman primate (NHP) and assess maternal, placental and fetal responses to nanoparticle-mediated IGF1 treatment. Methods: Pregnant macaques underwent ultrasound-guided intraplacental injections of nanoparticles ( GFP- or IGF1- expressing plasmid under the control of the trophoblast-specific PLAC1 promoter complexed with a HPMA-DMEAMA co-polymer) at approximately gestational day 100 (term = 165 days). Fetectomy was performed 24 h ( GFP ; n =1), 48 h ( IGF1 ; n = 3) or 10 days ( IGF1 ; n = 3) after nanoparticle delivery. Routine pathological assessment was performed on biopsied maternal tissues, and placental and fetal tissues. Maternal blood was analyzed for complete blood count (CBC), immunomodulatory proteins and growth factors, progesterone (P4) and estradiol (E2). Placental ERK/AKT/mTOR signaling was assessed using western blot and qPCR. Findings: Fluorescent microscopy and in situ hybridization confirmed placental uptake and transgene expression in villous syncytiotrophoblast. No off-target expression was observed in maternal and fetal tissues. Histopathological assessment of the placenta recorded observations not necessarily related to the IGF1 nanoparticle treatment. In maternal blood, CBCs, P4 and E2 remained within the normal range for pregnant macaques across the treatment period. Changes to placental ERK and AKT signaling at 48 h and 10 d after IGF1 nanoparticle treatment indicated an upregulation in placental homeostatic mechanisms to prevent over activity in the normal pregnancy environment. Interpretation: Maternal toxicity profile analysis and lack of adverse reaction to nanoparticle-mediated IGF1 treatment, combined with changes in placental signaling to maintain homeostasis indicates no deleterious impact of treatment. Funding: National Institutes of Health, and Wisconsin National Primate Research Center.

Interventions for placental insufficiency and fetal growth restriction.

Pregnancy complications adversely impact both mother and/or fetus throughout the lifespan. Fetal growth restriction (FGR) occurs when a fetus fails to reach their intrauterine potential for growth, it is the second highest leading cause of infant mortality, and leads to increased risk of developing non-communicable diseases in later life due 'fetal programming'. Abnormal placental development, growth and/or function underlies approximately 75% of FGR cases and there is currently no treatment save delivery, often prematurely. We previously demonstrated in a murine model of FGR that nanoparticle mediated, intra-placental human IGF-1 gene therapy maintains normal fetal growth. Multiple models of FGR currently exist reflecting the etiologies of human FGR and have been used by us and others to investigate the development of in utero therapeutics as discussed here. In addition to the in vivo models discussed herein, utilizing human models including in vitro (Choriocarcinoma cell lines and primary trophoblasts) and ex vivo (term villous fragments and placenta cotyledon perfusion) we have demonstrated robust nanoparticle uptake, transgene expression, nutrient transporter regulation without transfer to the fetus. For translational gene therapy application in the human placenta, there are multiple avenues that require investigation including syncytial uptake from the maternal circulation, transgene expression, functionality and longevity of treatment, impact of treatment on the mother and developing fetus. The potential impact of treating the placenta during gestation is high, wide-ranging across pregnancy complications, and may offer reduced risk of developing associated cardio-metabolic diseases in later life impacting at both an individual and societal level.

Suppression of SIN3A by miR-183 Promotes Breast Cancer Metastasis.

Recent work has established that SWI-independent-3 (SIN3) chromatin modification complexes play key roles in cancer progression. We previously demonstrated that knockdown of SIN3A expression promotes human breast cancer cell invasion and metastasis; however, the levels of SIN3A in patient breast carcinoma are not known. We therefore examined SIN3A mRNA and protein in patient tissues and determined that SIN3A expression is lower in breast carcinoma relative to normal breast. Given the 3'-untranslated region (UTR) of SIN3A has several conserved binding sites for oncogenic miRNA, we hypothesized that SIN3A is targeted by miRNA and found that ectopic miR-183 results in decreased SIN3A in breast carcinoma cell lines. Functionally, we demonstrate that miR-183 promotes breast cancer cell migration and invasion in a SIN3A-dependent manner and ectopic miR-183 promotes metastasis in vivo. Patients with breast cancer with high levels of miR-183 and low levels of SIN3A have the shortest overall survival. Given the critical link between metastasis and survival in patients with breast cancer, it is of utmost importance to identify clinically relevant genes involved in metastasis. Here, we report for the first time the aberrant expression of the putative metastasis suppressing gene SIN3A in human breast cancers and propose a mechanism of SIN3A suppression by miR-183. IMPLICATIONS: SIN3A expression is decreased in metastatic breast cancer in part due to miR-183.

Placental treatment with insulin-like growth factor 1 via nanoparticle differentially impacts vascular remodeling factors in guinea pig sub-placenta/decidua.

Clinically, fetal growth restriction (FGR) is only detectable in later gestation, despite pathophysiological establishment likely earlier in pregnancy. Additionally, there are no effective in utero treatment options for FGR. We have developed a nanoparticle to deliver human insulin-like 1 growth factor (hIGF-1) in a trophoblast-specific manner which results in increased expression of hIGF-1. IGF-1 signaling in the placenta regulates multiple developmental processes including trophoblast invasion and maternal vascular remodeling, both of which can be diminished in the FGR placenta. We aimed to determine the effects of short-term hIGF-1 nanoparticle treatment on sub-placenta/decidua trophoblast signaling mechanisms in FGR and under normal growth conditions. Using the guinea pig maternal nutrient restriction (MNR) model of FGR, ultrasound-guided, intra-placenta injections of hIGF-1 nanoparticle were performed at gestational day 30-33, and dams sacrificed 5 days later. Sub-placenta/decidua tissue was separated from placenta for further analyses. Western blot was used to analyze protein expression of ERK/AKT/mTOR signaling proteins (phospho-Erk (pERK), phospho-Akt (pAKT), raptor, rictor and deptor). qPCR was used to analyze gene expression of vascular/remodeling factors [vascular endothelial growth factor (Vegf), placenta growth factor (Pgf), platelet-derived growth factor (Pdgf)) and tight junction/adhesion proteins (claudin 5 (Cldn5), p-glycoprotein (Abcb1), occludin (Ocln) and tight junction protein 1 (Zo1)]. MNR reduced expression of pERK, PdgfB and Cldn5, and increased expression of Ocln and Zo1 in the sub-placenta/decidua. In MNR + hIGF1 nanoparticle sub-placenta/decidua, expression of PdgfB, Ocln and Zo1 was normalized, whilst pAkt, VegfB, Vegf receptor 1 and PdgfB receptor were increased compared to MNR. In contrast, hIGF-1 nanoparticle treatment of normal placentas reduced expression of pERK, raptor and increased expression of the mTOR inhibitor deptor. This was associated with reduced expression of VegfA, Plgf, and PdgfB. Here we have shown that the impact of hIGF-1 nanoparticle treatment is dependent on pregnancy environment. Under MNR/FGR, hIGF-1 nanoparticle treatment triggers increased expression of growth factors and normalization of EMT factors. However, under normal conditions, the response of the placenta is to decrease AKT/mTOR signaling and growth factor expression to achieve homeostasis.