K Abi-Nader

Long-term increase in uterine blood flow is achieved by local overexpression of VEGF-A 165 in the uterine arteries of pregnant sheep

Increasing uterine artery blood flow (UABF) may benefit fetal growth restriction where impaired uteroplacental perfusion prevails. Based on previous short-term results, we examined the long-term effects of adenovirus vector-mediated overexpression of vascular endothelial growth factor-A165 (VEGF-A165) in the uterine artery (UtA). Transit-time flow probes were implanted around both UtAs of mid-gestation pregnant sheep (n=11) to measure UABF. A carotid artery catheter was inserted to measure maternal or fetal hemodynamics. Baseline UABF was measured over 3 days, before injection of adenovirus vector (5 × 1011 particles) encoding the VEGF-A165 gene (Ad.VEGF-A165) into one UtA and a reporter β-galactosidase gene (Ad.LacZ) contralaterally. UABF was then measured daily until term. At 4 weeks post injection, the increase in UABF was significantly higher in Ad.VEGF-A165 compared with Ad.LacZ-transduced UtAs (36.53% vs 20.08%, P=0.02). There was no significant effect on maternal and fetal blood pressure. Organ bath studies showed significantly lesser vasoconstriction (Emax 154.1 vs 184.7, P<0.001), whereas immunohistochemistry demonstrated a significantly increased number of adventitial blood vessels (140 vs 91, n=26, P<0.05) following Ad.VEGF-A165 transduction. Local overexpression of VEGF-A165 in the UtAs of pregnant mid-gestation sheep leads to a sustained long-term increase in UABF, which may be explained by neovascularization and altered vascular reactivity.

Recombinant adeno-associated virus-mediated in utero gene transfer gives therapeutic transgene expression in the sheep.

Somatic in utero gene therapy aims to treat congenital diseases where pathology develops in perinatal life, thereby preventing permanent damage. The aim of this study was to determine whether delivery of self-complementary (sc) adeno-associated virus (AAV) vector in utero would provide therapeutic long-term transgene expression in a large animal model. We performed ultrasound-guided intraperitoneal injection of scAAV2/8-LP1-human Factor IX (hFIX)co (1 × 10(12) vector genomes/kg) in early (n = 4) or late (n = 2) gestation fetal sheep. The highest mean hFIX levels were detected 3 weeks after injection in late gestation (2,055 and 1,687.5 ng/ml, n = 2) and 3 days after injection in early gestation (435 ng/ml, n = 1). Plasma hFIX levels then dropped as fetal liver and lamb weights increased, although low levels were detected 6 months after late gestation injection (75 and 52.5 ng/ml, n = 2). The highest vector levels were detected in the fetal liver and other peritoneal organs; no vector was present in fetal gonads. hFIX mRNA was detectable only in hepatic tissues after early and late gestation injection. Liver function tests and bile acid levels were normal up to a year postnatal; there was no evidence of liver pathology. No functional antibodies to hFIX protein or AAV vector were detectable, although lambs mounted an antibody response after injection of hFIX protein and Freunds adjuvant. In conclusion, hFIX expression is detectable up to 6 months after delivery of scAAV vector to the fetal sheep using a clinically applicable method. This is the first study to show therapeutic long-term hFIX transgene expression after in utero gene transfer in a large animal model.

Local Over-Expression of VEGF-DΔNΔC in the Uterine Arteries of Pregnant Sheep Results in Long-Term Changes in Uterine Artery Contractility and Angiogenesis

Background The normal development of the uteroplacental circulation in pregnancy depends on angiogenic and vasodilatory factors such as vascular endothelial growth factor (VEGF). Reduced uterine artery blood flow (UABF) is a common cause of fetal growth restriction; abnormalities in angiogenic factors are implicated. Previously we showed that adenovirus (Ad)-mediated VEGF-A165 expression in the pregnant sheep uterine artery (UtA) increased nitric oxide synthase (NOS) expression, altered vascular reactivity and increased UABF. VEGF-D is a VEGF family member that promotes angiogenesis and vasodilatation but, in contrast to VEGF-A, does not increase vascular permeability. Here we examined the effect of Ad.VEGF-DΔNΔC vector encoding a fully processed form of VEGF-D, on the uteroplacental circulation. Methods UtA transit-time flow probes and carotid artery catheters were implanted in mid-gestation pregnant sheep (n = 5) to measure baseline UABF and maternal haemodynamics respectively. 7–14 days later, after injection of Ad.VEGF-DΔNΔC vector (5×1011 particles) into one UtA and an Ad vector encoding β-galactosidase (Ad.LacZ) contralaterally, UABF was measured daily until scheduled post-mortem examination at term. UtAs were assessed for vascular reactivity, NOS expression and endothelial cell proliferation; NOS expression was studied in ex vivo transduced UtA endothelial cells (UAECs). Results At 4 weeks post-injection, Ad.VEGF-DΔNΔC treated UtAs showed significantly lesser vasoconstriction (Emax144.0 v/s 184.2, p = 0.002). There was a tendency to higher UABF in Ad.VEGF-DΔNΔC compared to Ad.LacZ transduced UtAs (50.58% v/s 26.94%, p = 0.152). There was no significant effect on maternal haemodynamics. An increased number of proliferating endothelial cells and adventitial blood vessels were observed in immunohistochemistry. Ad.VEGF-DΔNΔC expression in cultured UAECs upregulated eNOS and iNOS expression. Conclusions Local over-expression of VEGF-DΔNΔC in the UtAs of pregnant mid-gestation sheep reduced vasoconstriction, promoted endothelial cell proliferation and showed a trend towards increased UABF. Studies in cultured UAECs indicate that VEGF-DΔNΔC may act in part through upregulation of eNOS and iNOS.

Doppler Ultrasonography for the Noninvasive Measurement of Uterine Artery Volume Blood Flow Through Gestation in the Pregnant Sheep

Accurate noninvasive quantification of volume blood flow in the uterine arteries (UtAs) would have clinical and research benefits. We evaluated the correlation and agreement between uterine artery volume blood flow (UtABF) as calculated (cUtABF) from color/pulsed-wave Doppler acquisitions and that measured (mUtABF) by bilateral perivascular transit-time flow probes in 6 pregnant sheep at 2 gestational ages. Out of 22 Doppler acquisitions, 19 were successful. The overall correlation between cUtABF and mUtABF was 0.55 (n = 19, P = .01). Calculated UtABF and mUtABF were significantly correlated in late gestation (n = 11, r = 0.71, P = .01) but not at mid-gestation (n = 8, r = .02, P = .96). By Bland-Altman analysis, the mean cUtABF/mUtABF was 1.15 with 95% limit of agreement (-0.26 to 2.56), similar to results previously achieved using power/pulsed-wave Doppler. Despite the acceptable correlation, the limits of agreement between Doppler and transit-time flow probe measurements remain wide. This makes Doppler ultrasonography less than a desirable method to quantify UtABF in studies where accurate quantification is required.

Monitoring for Potential Adverse Effects of Prenatal Gene Therapy: Use of Large Animal Models with Relevance to Human Application

Safety is an absolute prerequisite for introducing any new therapy, and the need to monitor the consequences of administration of both vector and transgene to the fetus is particularly important. The unique features of fetal development that make it an attractive target for gene therapy, such as its immature immune system and rapidly dividing populations of stem cells, also mean that small perturbations in pregnancy can have significant short- and long-term consequences. Certain features of the viral vectors used, the product of the delivered gene, and sometimes the invasive techniques necessary to deliver the construct to the fetus in utero have the potential to do harm. An important goal of prenatal gene therapy research is to develop clinically relevant techniques that could be applied to cure or ameliorate human disease in utero on large animal models such as sheep or nonhuman primates. Equally important is the use of these models to monitor for potential adverse effects of such interventions. These large animal models provide good representation of individual patient-based investigations. However, analyses that require defined genetic backgrounds, high throughput, defined variability and statistical analyses, e.g. for initial studies on teratogenic and oncogenic effects, are best performed on larger groups of small animals, in particular mice. This chapter gives an overview of the potential adverse effects in relation to prenatal gene therapy and describes the techniques that can be used experimentally in a large animal model to monitor the potential adverse consequences of prenatal gene therapy, with relevance to clinical application. The sheep model is particularly useful to allow serial monitoring of fetal growth and well-being after delivery of prenatal gene therapy. It is also amenable to serially sampling using minimally invasive and clinically relevant techniques such as ultrasound-guided blood sampling. For more invasive long-term monitoring, we describe telemetric techniques to measure the haemodynamics of the mother or fetus, for example, that interferes minimally with normal animal behaviour. Implanted catheters can also be used for serial fetal blood sampling during gestation. Finally, we describe methods to monitor events around birth and long-term neonatal follow-up that are important when considering human translation of this therapy.

Animal models for prenatal gene therapy: the sheep model.

Large animal experiments are vital in the field of prenatal gene therapy, to allow translation from small animals into man. Sheep provide many advantages for such experiments. They have been widely used in research into fetal physiology and pregnancy and the sheep fetus is a similar size to that in the human. Sheep are tolerant to in utero manipulations such as fetoscopy or even hysterotomy, and they are cheaper and easier to maintain than non-human primates. In this chapter, we describe the animal husbandry involved in generating time-mated sheep pregnancies, the large number of injection routes in the fetus that can be achieved using ultrasound or fetoscopic-guided injection, and laparotomy when these more minimally invasive routes of injection are not feasible.

Dichorionic Triamniotic Triplet Pregnancy Complicated by Acardius Acormus

This report describes an acardiac fetus of the acormus phenotype in a triplet pregnancy. The diagnosis was confirmed at 15 weeks. In the absence of signs of heart failure in the co-fetus the pregnancy was managed conservatively. The pregnancy was complicated by preterm labour and the fetuses were delivered at 26+5 weeks. The prenatal diagnosis of the acormus phenotype with a well-developed cephalic pole is extremely rare and has never been described antenatally in a higher order multiple pregnancy. We suggest that this rare acardiac fetus phenotype may have a different pathophysiology than those of other phenotypes. The report also summarizes the perinatal outcomes of triplet pregnancies complicated by an acardiac fetus, where the median gestational age at delivery is 26–27 weeks, and discusses the possible therapeutic interventions.

Prenatal Gene Therapy for the Early Treatment of Genetic Disorders

Prenatal gene therapy aims to deliver genes to cells and tissues early in prenatal life, allowing the correction of a genetic defect before long-term tissue damage has occurred. In contrast to postnatal gene therapy, prenatal application has a number of advantages, including targeting genes in a large population of dividing stem cells, and the smaller fetal size, which allows a higher vector-to-target cell ratio to be achieved. Early gestational delivery may result in the fetus developing immune tolerance to the transgenic protein, which would allow postnatal repeat of vector administration if needed. Recent advances in vector design and stem cell research have benefited this potential treatment. Although still in the preclinical stage, proof-of-principle studies in animal models of congenital disease, such as the hemophilic mouse, have shown the potential of prenatal gene therapy. Investigators have devised delivery strategies in large animals that could be used clinically to apply gene therapy to a huma...

Bilateral neck cysts as an isolated sonographic finding in the antenatal detection of fetal aneuploidy: a case report.

Isolated fetal lateral neck cysts can represent a cystic hygroma or a developmental remnant cyst. In the absence of an increased nuchal translucency or associated malformations the risk of aneuploidy has been considered negligible. Still, dysmorphology in aneuploid fetuses might not be evident except at a later stage. We report on a case of isolated fetal bilateral neck cysts where aneuploidy was suspected and confirmed despite the lack of associated morphologic abnormalities.

Fetal Muscle Gene Therapy/Gene Delivery in Large Animals

Gene delivery to the fetal muscles is a potential strategy for the early treatment of muscular dystrophies. In utero muscle gene therapy can also be used to treat other genetic disorders such as hemophilia, where the missing clotting proteins may be secreted from the treated muscle. In the past few years, studies in small animal models have raised the hopes that a phenotypic cure can be obtained after fetal application of gene therapy. Studies of efficacy and safety in large animals are, however, essential before clinical application can be considered in the human fetus. For this reason, the development of clinically applicable strategies for the delivery of gene therapy to the fetal muscles is of prime importance. In this chapter, we describe the protocols for in utero ultrasound-guided gene delivery to the ovine fetal muscle in early gestation. In particular, procedures to inject skeletal muscle groups such as the thigh and thoracic musculature and targeting the diaphragm in the fetus are described in detail.