Percutaneous Ultrasound-Guided Injection of the Trachea in Fetal Sheep: A Novel Technique to Target the Fetal Airways

Abstract

Objective: To access the fetal airways percutaneously using ultrasound-guided injection of the fetal trachea in sheep. Methods: Adenoviral gene therapy vectors and transduction-enhancing agents were delivered to the trachea via a needle inserted through the thorax or the neck of late-gestation (0.9 term, n = 3) or mid-gestation (0.5–0.8 term, n = 18) fetal sheep using ultrasound guidance. Results: Injection of the trachea in the fetal thorax was successful in 16 out of 18 fetuses and achieved at the first attempt in 9 fetuses within 12 min [mean 7 min and 31 s ± (SD) 3 min and 4 s]. Survival was 100%. Injecting the trachea in the neck was less successful. Conclusions: The fetal trachea of the sheep can be safely accessed by percutaneous ultrasound-guided injection to deliver vectors directly to the fetal airways for gene therapy. It may also enable tracheal occlusion for the antenatal treatment of congenital diaphragmatic hernia without the need for endoscopy or open surgery.

Widespread and efficient marker gene expression in the airway epithelia of fetal sheep after minimally invasive tracheal application of recombinant adenovirus in utero

“…Although trans-thoracic access to the fetal sheep trachea was possible as early as day 81 and up to day 138 of gestation, we found that delivery was best achieved between days 81 and 110. 18 A dose of 1 Â 10 12 adenolacZ vector particles was applied and low-level, dispersed b-galactosidase expression limited to the peripheral airways or parenchyma was detected 48 h after injection ( Figure 2, panel 1a). To obtain a more quantifiable measure of gene transfer following this procedure, two defined 5 mm thick cross-sections of the lung were used for determination of transgenic b-galactosidase activity by ELISA.…”

“…Optimization of the technique for percutaneous ultrasound-guided injection of the trachea, which did result in some initial fetal mortality, has been described previously. 18 No serious morphological alterations were observed at post-mortem analysis following in utero tracheal delivery of adenovirus complexed with DEAE dextran or pretreatment of airways with Na-caprate.…”

“…18 The needle was passed between the second and third or third and fourth ribs of the fetal chest, via the lung parenchyma, into the trachea just proximal to the carina. Correct placement of the needle was confirmed by withdrawal of tracheal fluid and the vector was injected over 2 min.…”

Gene transfection into fetal sheep airways in utero using guanidinium‐cholesterol cationic lipids

“…In these ongoing experiments, tracheal occlusion is actually performed via an occlusion balloon as previously reported by others [14]. In addition, as suggested by recent work on adenoviral delivery to early gestation fetal sheep, ultrasound-based techniques may in the future enable percutaneous tracheal occlusion without the need for endoscopy or open surgery [40,41]. Accordingly, a recent clinical trial has demonstrated that fetal tracheal occlusion for CDH clearly requires the development of minimally invasive techniques in order to improve the results in terms of fetal mortality and morbidity [42].…”

Gene Editing and Genetic Lung Disease. Basic Research Meets Therapeutic Application

“…Therapeutic intervention in the fetal period may induce immune tolerance and improve efficiency. Finally, prenatal application is feasible via the intraamniotic, vitelline vein, or by fetal intratracheal injection (48)(49)(50)(51). The presence of fetal breathing movements combined with direct contact between the developing airways and amniotic fluid suggests that CRISPR/Cas9 administered via the intraamniotic route could reach the lung epithelium.…”