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^1,2,3,4,5. Using pigs as a large animal model, we have refined a protocol to achieve widespread airway distribution following intratracheal aerosol delivery.

Here we demonstrate how to achieve efficient viral vector delivery to a pig lung through aerosolization. Conceptually, topical delivery of a vector encoding a therapeutic transgene to the lung is simple. However, in practice, achieving efficient delivery is a challenge. Important considerations include the viral vector, the appropriate vehicle for the vector, and the aerosolization method. In general, devices for generating airborne vectors can be categorized as follows: aerosolizing catheters, atomizers, and nebulizers. All devices convert liquids into particles small enough for respiration. Aerosolizing catheters convert liquids into particles at expulsion. For these studies, we use a syringe-mounted aerosolizing catheter named a microsprayer. We selected a microsprayer as our aerosolization device in part because of its ease of use and because of its ability to effectively aerosolize a viral vector in a particle size that can reach all areas of the lung. We quantified droplet geometric size by laser diffraction and obtained consistent measurements of 15-16 µm for each droplet. The microsprayer works by generating an aerosol at its tip that results from the force generated by depressing a syringe plunger. We validated this delivery method for both adenoviral (Ad)- and adeno-associated virus (AAV)-based viral vectors^6,7.

Alternatively, there are aerosolizing catheter devices that utilize pressurized delivery through compressed air. Particle sizes as small as 4-8 µm may be possible with pressurized delivery. Such a device was used to aerosolize helper-dependent adenovirus vectors to rabbit airways^8,9 and Sendai virus vectors to sheep¹⁰. Atomizers are a type of aerosolizing catheters that deliver large sized particles (~30-90 µm diameter). We have observed that this type of atomizer is effective for delivering multiple viral vectors, including lentiviral vectors, especially when formulated with a viscoelastic material such as methylcellulose¹¹. Nebulizers first convert the liquid into a mist that is passively inhaled. Using this strategy, a plasmid-based vector was delivered to the airways of CF patients in a phase IIB gene therapy trial¹². Nebulization requires a large volume of concentrated material and is therefore the least economical option for delivery of viral vectors.

Prior to developing this protocol, we tested multiple different delivery methods in newborn pigs. We evaluated localized delivery via a pediatric bronchoscope lined with either a PE20 catheter delivered as a bolus liquid dose, or through a drug infusion balloon¹³. Additionally, we tested an atomizer¹⁴ and a pressurized aerosolizing catheter (unpublished). The pressurized aerosolizing catheter delivery was effective but required extra equipment and the pressurized delivery occasionally resulted in injury to pig tracheas. Based on ease of use and reproducibility, we now routinely opt for the syringe-mounted microsprayer for delivery of encapsidated viral vectors such as adenoviral and adeno-associated viral vectors. The atomizer gives the most comparable lung expression to the microsprayer without needing to pass through an endotracheal tube. Although our focus has been on developing a delivery method for efficient lung gene transfer to correct cystic fibrosis, this method could be adapted for other applications. The aerosolization device and droplet size may play an important role in the efficiency and distribution of vector mediated transgene expression. Here, we focus on the procedure of intubation in newborn pigs and passing an aerosolizing catheter through an endotracheal (ET) tube to deliver vector.

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 and mark the base of the microsprayer when the tip exits the ET tube by ~1 mm. This will serve as a guide for how far to insert the microsprayer into the ET tube once it is placed in the animal's airway.
   NOTE: Not all ET tubes are the same length, so this step should be repeated for every new ET tube used in a procedure.
4. Remove the microsprayer from the ET tube and screw the microsprayer onto to a luer locking syringe loaded with 1-2 mL of viral vector. Set aside.
   NOTE: A test spray through the microsprayer is recommended prior to setup. Prefilling the spray nozzle with viral vector is not necessary.
5. Insert a stylet into the ET tube to support intubation.
6. Pigs will be anesthetized using 2-4% isoflurane. Assemble an isoflurane vaporizer.
   1. Connect an O₂ tank with a pressure regulator and flowmeter to the vaporizer. Connect the vaporizer through tubing to deliver the isoflurane through an anesthesia mask and an anesthesia gas filter canister to collect the waste anesthesia gas from the operating room environment.
7. Within the procedure area, arrange a laryngoscope with a 4 in elongated blade, the microsprayer with the syringe containing the vector, and an ET tube lined with a stylet. Precoat the ET tube tip with lubricating jelly.

2. Sedate the pigs.

1. Use a pulse oximeter to measure the pig's oxygen saturation and the heart rate. Place a wraparound SpO₂ sensor around the pig's hind leg and ensure the readings register on the pulse oximeter. Record the pre-anesthetic reading.
   NOTE: Animals must fast prior to sedation to prevent aspiration during delivery.
2. Turn on the O₂ tank (flowmeter set to 2 L/min) and the isoflurane vaporizer to begin the flow to the anesthesia mask.
3. Place the anesthesia mask over the pig's snout and hold the pig until sedated. This may take approximately 4-5 min but will vary depending on the age and weight of the animal. Begin by holding the pig during the initial stages of anesthesia. Once the pig is sedated, lay it on the prepared procedure space (i.e., the underpad over a heating pad). Confirm anesthesia by testing the pedal reflex.
   NOTE: The animal should never be left unattended.
4. Record the rectal temperature and the respiratory rate.
5. Continue the procedural monitoring every 15 min throughout sedation (i.e., SpO₂, heart rate, temperature, and respiratory rate).

3. Intubate the sedated pigs with an endotracheal tube.

1. Confirm that the ET tube with the stylet has been lightly coated with lubricating jelly to facilitate intubation (step 1.6).
2. Remove the anesthesia mask from the pig and turn off the flow of isoflurane.
3. Lay the pig supine on the procedure space and visualize the larynx using a laryngoscope.
4. Pass a 2.0 mm ET tube through the vocal folds of the larynx and into the trachea (Supplemental Movie 1). If the pig is properly intubated, the SpO₂ levels will start to decline.
   NOTE: The exact ET tube placement will vary depending on the size of the animal. Placement can vary from just beyond the larynx for larger animals (~3-4 kg) to near the carina for smaller animals (~0.8-1 kg). In small animals, there is risk of one side intubation and trimming 3-5 cm from the ET tube may be warranted.
5. Remove the stylet.

4. Aerosolize the viral vector using the microsprayer.

1. Pass the microsprayer connected to the viral vector-containing syringe through the ET tube until reaching the mark at the base of the microsprayer.
2. Spray the solution intratracheally by pressing the syringe plunger with a firm and consistent force to generate a mist. This will take approximately 3-4 s.
   NOTE: The appropriate pressure should be practiced beforehand. Too little pressure will result in a stream instead of a spray.
   NOTE: For our studies, we limited the volume delivered to ~1 mL/kg.
3. A post-spray "air chaser" of ~500 µL will help ensure complete delivery of the vector from the syringe and nozzle.
4. Gently remove the ET tube and microsprayer from the intratracheal intubation at the same time. Successful delivery will typically result in the sound of crackles when breathing.
   NOTE: A typical procedure time from initiated anesthesia time to extubation is 10-15 min.

5. Monitor the pigs as they come out of sedation.

NOTE: Apnea is a common response to intubation in newborn pigs. Sporadic breathing may last 2-3 min. Gentle chest compressions can help facilitate normal breathing.

1. Monitor SpO₂ levels until they return to 95-100% then remove the SpO₂ sensor from the pig's hind leg.
2. Continue with the postprocedural monitoring every 15 min until the pig is alert, sternal, and walking. Typically, pigs will recover within 15 min.

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