Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery

Podsumowanie

Salivary glands have been proposed as a tissue target site for gene therapy, especially in the area of vaccination by gene transfer. We demonstrate gene delivery in a non-human primate model utilizing retrograde parotid infusion.

Streszczenie

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of secreting proteins into the bloodstream and are easily accessible, making them potentially superior tissue sites for replacement hormone production or vaccination by gene transfer. Suggested methods for gene delivery include transcutaneous injection and retrograde infusion through salivary ducts. We demonstrate how to perform Retrograde Salivary Gland Infusion (RSGI) in non-human primates. We describe the important anatomic landmarks including identification of the parotid papilla, an atraumatic method of cannulating and sealing Stensen's Duct utilizing basic dental tools, polyethylene tubing, and cyanoacrylate, and the appropriate rate of infusion. While this is the least traumatic method of delivery, the method is still limited by the volume able to be delivered (<0.5 mL) and the potential for trauma to the duct and gland. We demonstrate using fluoroscopy that an infusate can be fully delivered into the gland, and further demonstrate by immunohistochemistry the transduction of a typical vector and expression of the delivered gene.

Wprowadzenie

While salivary glands are well known for their exocrine production of saliva, researchers have long recognized their ability to secrete proteins directly into the bloodstream¹, making them a potential target for gene therapy for systemic administration, such as replacement hormones or antibody production. In fact, salivary glands offer several advantages over other tissue targets, such as the inherent ability to produce proteins for secretion (a property muscles lack), heavy encapsulation that can limit vector diffusion, and well-differentiated tissue providing stability for non-integrating vectors. Furthermore, in the event of a serious adverse event, salivary glands are not critical for life and can be surgically removed. While not immediately intuitive, parotid glands are also easily accessible from the mouth through their main excretory duct, Stensen's Duct².

Given the advantages of salivary tissue for gene therapy, there is increasing interest in exploring this tissue target. Numerous studies have already been performed in rodent, canine, and non-human primate models and at least one human clinical trial is underway^3,4,5. To further explore and develop the utility of this tissue target for gene therapy purposes, more non-human primate studies will need to be performed. This paper describes a method for accessing the parotid glands through Stensen's Duct to deliver a vectored gene for transduction in the non-human primate model. To visibly demonstrate the delivery of the infusate and the anatomy of the duct as it enters the gland, fluoroscopy using radiocontrast was performed. To demonstrate successful transduction of a vector, an Adenovirus serotype 5 (Ad5) vectored egfp gene was used. Ad5 is a well-described vector capable of transducing salivary tissue. Although it is too immunogenic for ultimate clinical use, an Ad5 vector was chosen for this demonstration study to assure efficient transduction. Evaluating Enhanced Green Fluorescent Protein (EGFP) production is a well-described method to demonstrate successful transcription and translation of a vectored gene following transduction and was done here.

Protokół

All procedures were performed at Wake Forest School of Medicine Clarkson Campus for animal studies. The Institutional Animal Care and Use Committee (IACUC) was consulted for ethical considerations and details of the procedures was submitted for review. Wake Forest IACUC approved our study protocol and all procedures were done under IACUC approved protocol #A17-147.

1. Preparing the infusion device

1. Cut size 10 Polyethylene Tube (PET10) into 25 cm lengths using a pair of scissors.
2. Mark PET10 at 1 cm and 2 cm from one end using a black marker.
3. Prefill 0.5 mL of Ad5-EGFP solution (10⁹ viral particles/mL) into a 1 mL (tuberculin) syringe.
4. Slide non-marked end of PET10 tube over 29-31 G needle attached to a syringe. It is generally easier to perform this task under magnification.
5. Infuse the solution into the PET10 until tube is completely full (visible drop at free end).
6. Use full standard PPE, including surgical scrubs, long sleeved gown, impermeable gloves, surgical mask, face shield, hair bonnet, and shoe covers.

2. Preparing the animal

NOTE: Cynomolgus macaques were used for the video demonstration. The anatomy of other non-human and human primates is very similar, and the protocol should be translatable to other species.

1. Inject subcutaneously 0.05 mg/kg of atropine 15 min prior to the procedure to minimize salivary secretions and optimize distribution and retention of the infusate.
2. Provide anesthesia using 5 mL syringes with intramuscular ketamine/midazolam (10-15 mg/kg of ketamine and 0.01-0.05 mg/kg of midazolam). Confirm proper anesthesia when the sedated animal becomes unconscious and is unable to react to stimuli.

3. Performing the procedure

1. Use oral retractors to brace open mouth.
   1. Place the rubber pad of one end of the retractor on the hard palate behind the upper teeth on the side of the mouth opposite to the gland that will be infused. Place the rubber pad of the other end on the lower canine on the same side as upper retractor. Gently allow the spring action of the retractor to expand and open the mouth.
2. Identify the parotid papilla, the opening of Stensen's Duct, on the posterior cheek, adjacent to the upper 2^nd molar. This is best visualized using dental loops for magnification.
3. Gently dilate the parotid papilla with the point of the conical dilator. It is best to place the point of the dilator into the center or opening of the papilla and then gently rotate it back and forth. The point should slowly enter the papilla and dilate it over approximately 20 - 30 s of gentle rotating.
4. Insert the PET10 tubing into the dilated parotid papilla. This is best achieved by holding the marked end of the PET10 tube with tweezers approximately 0.5 cm from the distal end and gently inserting the tip of the tube into the dilated papilla.
   1. Gently advance the tube, which is often facilitated by small rotating movements to help the tube slide, followed by readjustment of the tweezers 0.5 cm proximal to the previous grip. Repeat this until the 2 cm mark reaches the parotid papilla.
5. Apply cyanoacrylate on the cheek around the papilla and the inserted tube and wait for it to dry (no specific amount recorded, just enough to seal the entrance of Stensen's duct papilla). This typically takes less than a minute and helps seal the parotid papilla and reduce spillage of infusate back in the oral cavity.
6. Slowly push the syringe content over 5 min at a rate of 100 µL/min. This slow infusion rate minimizes risk of duct injury due to sudden increase in intra-ductal pressure.
7. Leave PET10 in place for at least 5 min after infusion is complete. Keep the duct sealed and allow the infusate to remain in the parotid gland.
8. Remove PET10 with gentle traction. The cyanoacrylate will pull free with the tube.
9. Repeat steps 3.2 through 3.8 on the opposite side.
10. Slowly release oral retractors after both parotids have been infused and both PET10 tubes removed.
    NOTE: The whole procedure for both sides should take less than 30 min.

4. Post-procedural care

1. After infusion is completed and Stensen's duct decannulated, observe animals until anesthesia effect wears off (usually between 20-30 min post-procedure).
2. Offer the animal drinks and then food after they are fully awake and resume routine care.

Wyniki

Successful procedure, transduction and transcription
Figure 1 shows the parotid papilla adjacent to the 2^nd molar on the posterior superior cheek. The image also shows the correct placement of the mouth brace, one rubber end on the hard palate and the other rubber end on the ipsilateral canine. Figure 2 shows an image taken after successful cannulation of the parotid papilla at the 2 cm mark on the PET10. Fi...

Dyskusje

Here we describe a protocol of retrograde infusion into the parotid gland through Stensen's Duct. The methodology described offers guidance that can potentially be used by researchers exploring the utility of salivary tissue as a site for gene therapy and other applications.

There are multiple critical steps to ensure the success of the procedure. First and foremost, all the procedural steps should be completed gently. Forceful bracing of the mouth could result in mandibular subluxation. F...

Materiały

Name	Company	Catalog Number	Comments
500 µL U100 syringes with 30-gauge needles	Becton Dickinson	328466	fixed needle for less waste
Adhesive (e.g., Ethicon Dermabond)	Various		Cyanoacrylate adhesive to seal and keep the tubing in the duct during infusion.
Atropine injectable solution	Patterson Veterinary	07 869-6061	Atropine inj. 0.54 mg/mL
BD Ultra-Fine Insulin Syringes 30G	Walmart	N/A	Avilable in 0.5 mL and 1.0 mL sizes.
Cyanoacrylate (medical glue)	Ethicon	DNX12	Dermabond topical skin adhesive
Dental loops with light	Amazon (DDP)	B012M3IV80	Used to enhance visualization of Stensen's duct papilla
Infant Lacrimal Dilator	Surgipro	SPOI-137
Ketamine injectable solution	Patterson Veterinary	07-803-6637	Ketaset inj. 100 mg/mL
Lacrimal Dilator	Surgipro	SPOI-132	Used to dialate the Stensen's duct.
Midazolam injectable solution	Patterson Veterinary	07 890-6698	Midazolam inj. 5mg/mL
Pair of scissors	Amazon (DDP)	N/A	Used to cut PET10 tube
Polyethylene Tubing (PE-10)	Scientific Comodities, Inc	BB31695-PE/1	Tubing connecting the 30G syringe and inserted into the duct.
Q-tips	Walmart	N/A	Used to spread cyanoacrylate on the cheek
Size 10 Polyethylene Tube (PET 10)	Scientific Commodities	BB31695-PE/1	low density polyethylene tubing
Small Animal Mouth Opener	Amazon (DDP)	B01F3LVJXC	Used to keep the animal's mouth open.
Tweezers	Amazon (DDP)	N/A	Used to insert PET10 tube into Stenson's duct
Zinc Chloride	Sigma-Aldrich	7646-86-7	Included in plasmid DNA infusates