Intratracheal Aerosolization of Viral Vectors to Newborn Pig Airways

Summary

To maximize the potential benefits of pulmonary gene therapy, widespread and uniform topical delivery of a viral vector across the surface epithelium is an important goal. Here, we demonstrate an aerosolization technique using a microsprayer positioned intratracheally to deliver a viral vector to newborn pig airways.

Abstract

Gene therapy for airway diseases requires efficient delivery of nucleic acids to the intrapulmonary airways. In small animal models such as mice, gene delivery reagents are commonly delivered as a bolus dose. Routes of delivery may include either nasal sniffing or direct tracheal instillation. However, using a large animal model for preclinical applications is relevant for translation to human trials. Widespread and uniform distribution of transgene expression is critical for developing a successful lung gene therapy treatment. Aerosolizing viral vectors to the lungs of large animals, such as pigs or sheep, is a strategy to maximize gene transfer efficiency and results in greater airway distribution than a liquid bolus dose. Here we demonstrate a technique for direct aerosolization of a viral vector to the airways of newborn pigs. Briefly, a pig is anesthetized, intubated with an endotracheal tube, and a microsprayer is passed through the endotracheal tube. A syringe is used to push the vector through the microsprayer, resulting in a fine mist being released into the distal trachea. The microsprayer produces ~15-16 μm size particles that deposit across the proximal and distal regions of the lung. Using a microsprayer to deliver an adenoviral-based vector, we previously observed ~30-50% of surface epithelial cells transduced in both the large and small airways of newborn pigs.

Introduction

Gene transfer to the lung holds great potential for treating many genetic diseases, such as cystic fibrosis or alpha-1 antitrypsin deficiency. However, developing gene therapy approaches to successfully deliver genes of interest to the airways has been challenging. Animal models play a major role in driving innovation of viral vector design and delivery strategies to the intrapulmonary airways. Indeed, we and others have developed methods to overcome many gene delivery hurdles using large animal models. Many examples of delivery challenges have been previously reviewed^1,2,3

Protocol

All animal experiments performed following this protocol must be approved by the respective Institutional Animal Care and Use Committee (IACUC). All procedures described here were approved by the University of Iowa IACUC.

1. Prepare the procedure space and vector delivery materials.

1. Place a heating pad covered by a disposable underpad to warm the procedure area.
2. Set up the pulse oximeter to measure the heart rate and peripheral capillary oxygen saturation (SpO₂). Prepare a rectal thermometer by coating with a lubricating jelly.
3. Pass the microsprayer nozzle through a 2.0 mm inner diameter ET tube an....

Results

We previously validated this technique for delivering gene transfer vectors to pig lungs and showed widespread and uniform airway distribution following delivery of an adenoviral vector expressing green fluorescent protein (GFP)⁶. To assess transduction, all six lung lobes were separated into two to four segments. From each segment, tissue was designated for DNA or mRNA isolation and transduction was quantified by real-time PCR to detect the GFP sequence. GFP-posit.......

Discussion

Widespread airway distribution of a viral vector would help ensure the success of a gene therapy approach for treating pulmonary diseases. Here, we demonstrate an aerosolization technique that leads to whole lung expression of large and small pig airways. We describe the steps for sedating a pig, intubating with an ET tube, and aerosolizing a viral vector through the microsprayer aerosolization device. This technique is important as a preclinical approach to testing viral vector efficacy.

Ther.......

Materials

Name	Company	Catalog Number	Comments
Atomizer	Teleflex	MAD700	MADgic Laryngo-Tracheal Mucosal Atomization Device
Compressed Oxygen (O2) gas	Praxair		Also need pressure regulator and flowmeter
Disposable underpad	General stores
Endotracheal (ET) tube 2.0 mm I.D.	Teleflex Medical	5-10404	Hudson RCI; Sheridan Uncuffed
Fluorescent Dissecting Microscope	Leica	MDG41
Heating pad	General stores
Isoflurane	Pharmacy
Isoflurane F/Air Filter Canister	Vetamac Anesthesia	SKU VAD020
Isoflurane regulator (vaporizer)	Vetamac Anesthesia
Laryngoscope traditional set	DarvallVet	#8070	4" blade used for delivery
Leur locking syringe (3 ml)	General stores
Lubricating jelly	General stores
Microsprayer	PennCentury		Aerosolizing catheter; No longer available
Pressurized aerosolizing catheter	Trudell Medical Corporation		Aeroprobe; No longer available
Pulse oximeter	Pacific Medical Supply	UQNE4600
SpO2 sensor band	Hospital stores
Stylet	Hospital stores		5 Fr (1.7 mm O.D.)
Thermometer (Digital)	General stores
Veterinary anesthesia mask	Hospital stores
Viral vectors	University of Iowa Viral Vector Core		Adenoviral vector; fee for service