A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Summary

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Abstract

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome^1,2 or hyperoxic injuries of the neonatal lung³. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)⁴, genetic variants of surfactant deficiencies⁵ and α1-antitrypsin deficiency⁶.

Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies⁷.

In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep⁸, and even in a clinical setting⁹, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.

Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection^10-12, (ultrasound-guided) intrapulmonary injection^13,14 and intravenous administration into the yolk sac vessels^15,16 or umbilical vein¹⁷. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques¹⁸.

Protocol

1. Mating of Mice to Obtain Desired Pregnancy Stage

Time mate pregnant NMRI mice so that they are 18 days (E18) pregnant (total gestation E19.5) at the time of surgery. Prior to and after surgery they are housed in filter top cages at normal room temperature and normal daylight with free access to water and chow.

2. Fetal Intratracheal (I.T.) Injection (Figure 1)

1. First submit the pregnant NMRI mouse to general anesthesia with 1.5% isoflurane in a mixture of O₂ at 1.5 L/min. The level of isoflurane depends on the age and strain of the mouse, but in general isoflurane should be at a level that ....

Discussion

Critical steps

• The mouse strain we chose to work with is NMRI mice because they have an abundant number of pups (average litter size 14.4 ± 1.8, own data), tolerate interventions well and have good maternal characteristics.
• Placing the purse string through the uterine wall and the fetal membranes is a critical step as you only want to expose the fetal head and not the shoulders, otherwise repositioning is almost impossible without causing trauma.
• An optimal position of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NMRI mice	Janvier, Le Genest St Isle, France
Isoflurane	Isoba, Intervet / Schering-Plough Animal Health, Milton Keynes, UK
Prolene 6-0	Ethicon, Groot Bijgaarden, Belgium
Vicryl 5-0	Ethicon, Groot Bijgaarden, Belgium
50 μl Hamilton Glass Syringe, Model 1710.5 TLLX SYR	Hamilton, Reno, NV, USA	5495-20
30G sharp needle	Hamilton, Reno, NV, USA	7762-03
2% xylocaine	AstraZeneca, Zoetermeer, The Netherlands