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

Summary

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.

Abstract

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.

Introduction

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 advers....

Protocol

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/m....

Results

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. Figu.......

Discussion

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.......

Materials

List of materials used in this article

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