Name: retrograde parotid gland infusion through stensen's duct in a non-human primate for vectored gene delivery
Uploaded: 2021-08-12T14:00:00
Description: Watch this Scientific Journal Video about Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery at JoVE.com

The authors want to thank Mr. Cagney Gentry for his audiovisual support in filming the procedure. We also want to acknowledge the Hefner VA medical center for academic support in pursuit of this project.

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
<ol>
	<li>Cut size 10 Polyethylene Tube (PET10) into 25 cm lengths using a pair of scissors.</l...

<ol>
	<li>Isenman, L., Liebow, C., Rothman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretion+of+mammalian+digestive+enzymes+by+exocrine+glands.">The secretion of mammalian digestive enzymes by exocrine glands.</a> The American Journal of Physiology. 276, 223-232 (1999).</li><li>Perez, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+epithelial+cells:+An+unassuming+target+site+for+gene+therapeutics.">Salivary epithelial cells: An unassuming target site for gene therapeutics.</a> The International Journal of Biochemistry &amp; Cell Biology. 42, 773-777 (2010).</li><li>Kochel, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dengue+virus+serotype-1+DNA+vaccine+induces+virus+neutralizing+antibodies+and+provides+protection+from+viral+challenge+in+Aotus+monkeys.">A dengue virus serotype-1 DNA vaccine induces virus neutralizing antibodies and provides protection from viral challenge in Aotus monkeys.</a> Vaccine. 18, 3166-3173 (2000).</li><li>Ponzio, T. A., Sanders, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+salivary+gland+as+a+target+for+enhancing+immunization+response.">The salivary gland as a target for enhancing immunization response.</a> Tropical Diseases, Travel Medicine and Vaccines. 3, 4 (2017).</li><li>Baum, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+responses+to+adenoviral-mediated+transfer+of+the+aquaporin-1+cDNA+for+radiation-induced+salivary+hypofunction.">Early responses to adenoviral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 19403-19407 (2012).</li><li>Voutetakis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorting+of+Transgenic+Secretory+Proteins+in+Rhesus+Macaque+Parotid+Glands+After+Adenovirus-Mediated+Gene+Transfer.">Sorting of Transgenic Secretory Proteins in Rhesus Macaque Parotid Glands After Adenovirus-Mediated Gene Transfer.</a> Human Gene Therapy. 19, 1401-1405 (2008).</li><li>Niedzinski, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+systemic+transgene+expression+after+nonviral+salivary+gland+transfection+using+a+novel+endonuclease+inhibitor/DNA+formulation.">Enhanced systemic transgene expression after nonviral salivary gland transfection using a novel endonuclease inhibitor/DNA formulation.</a> Gene Therapy. 10, 2133-2138 (2003).</li><li>Niedzinski, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+Enhancement+of+Nonviral+Salivary+Gland+Transfection.">Zinc Enhancement of Nonviral Salivary Gland Transfection.</a> Molecular Therapy. 7, 396-400 (2003).</li><li>Samuni, Y., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+in+salivary+glands:+From+the+bench+to+the+clinic.">Gene delivery in salivary glands: From the bench to the clinic.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. , (2011).</li><li>Voutetakis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-Associated+Virus+Serotype+2-Mediated+Gene+Transfer+to+The+Parotid+Glands+of+Nonhuman+Primates.">Adeno-Associated Virus Serotype 2-Mediated Gene Transfer to The Parotid Glands of Nonhuman Primates.</a> Human Gene Therapy. 18, 142-150 (2007).</li></ol>

Here we describe a protocol of retrograde infusion into the parotid gland through Stensen&#39;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...

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&#39;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 underway3,4,5. 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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>500 &#181;L U100 syringes with 30-gauge needles</td>
			<td>Becton Dickinson</td>
			<td>328466</td>
			<td>fixed needle for less waste</td>
		</tr>
		<tr>
			<td>Adhesive (e.g., Ethicon Dermabond)</td>
			<td>Various</td>
			<td></td>
			<td>Cyanoacrylate adhesive to seal and keep the tubing in the duct during infusion.</td>
		</tr>
		<tr>
			<td>Atropine injectable solution</td>
			<td>Patterson Veterinary</td>
			<td>07 869-6061</td>
			<td>Atropine inj. 0.54 mg/mL</td>
		</tr>
		<tr>
			<td>BD Ultra-Fine Insulin Syringes 30G</td>
			<td>Walmart</td>
			<td>N/A</td>
			<td>Avilable in 0.5 mL and 1.0 mL sizes.</td>
		</tr>
		<tr>
			<td>Cyanoacrylate (medical glue)</td>
			<td>Ethicon</td>
			<td>DNX12</td>
			<td>Dermabond topical skin adhesive</td>
		</tr>
		<tr>
			<td>Dental loops with light</td>
			<td>Amazon (DDP)</td>
			<td>B012M3IV80</td>
			<td>Used to enhance visualization of Stensen&#39;s duct papilla</td>
		</tr>
		<tr>
			<td>Infant Lacrimal Dilator</td>
			<td>Surgipro</td>
			<td>SPOI-137</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine injectable solution</td>
			<td>Patterson Veterinary</td>
			<td>07-803-6637</td>
			<td>Ketaset inj. 100 mg/mL</td>
		</tr>
		<tr>
			<td>Lacrimal Dilator</td>
			<td>Surgipro</td>
			<td>SPOI-132</td>
			<td>Used to dialate the Stensen&#39;s duct.</td>
		</tr>
		<tr>
			<td>Midazolam injectable solution</td>
			<td>Patterson Veterinary</td>
			<td>07 890-6698</td>
			<td>Midazolam inj. 5mg/mL</td>
		</tr>
		<tr>
			<td>Pair of scissors</td>
			<td>Amazon (DDP)</td>
			<td>N/A</td>
			<td>Used to cut PET10 tube</td>
		</tr>
		<tr>
			<td>Polyethylene Tubing (PE-10)</td>
			<td>Scientific Comodities, Inc</td>
			<td>BB31695-PE/1</td>
			<td>Tubing connecting the 30G syringe and inserted into the duct.</td>
		</tr>
		<tr>
			<td>Q-tips</td>
			<td>Walmart</td>
			<td>N/A</td>
			<td>Used to spread cyanoacrylate on the cheek</td>
		</tr>
		<tr>
			<td>Size 10 Polyethylene Tube (PET 10)</td>
			<td>Scientific Commodities</td>
			<td>BB31695-PE/1</td>
			<td>low density polyethylene tubing</td>
		</tr>
		<tr>
			<td>Small Animal Mouth Opener</td>
			<td>Amazon (DDP)</td>
			<td>B01F3LVJXC</td>
			<td>Used to keep the animal&#39;s mouth open.</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Amazon (DDP)</td>
			<td>N/A</td>
			<td>Used to insert PET10 tube into Stenson&#39;s duct</td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7646-86-7</td>
			<td>Included in plasmid DNA infusates</td>
		</tr>
	</tbody>
</table>

retrograde parotid gland infusion through stensen's duct in a non-human primate for vectored gene delivery

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&#39;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 (&#60;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.

Successful procedure, transduction and transcription 
Figure 1 shows the parotid papilla adjacent to the 2nd 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...

Watch this Scientific Journal Video about Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery at JoVE.com

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

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.

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of ...

This protocol is important because it demonstrates the procedure of retrograde salivary gland infusion in detail. The procedure can be used for a variety of medical reasons, such as vectored immunoprophylaxis or gene therapy. This technique is easy to learn and reproduce.The authors have managed to perform this procedure successfully on multiple occasions in spite of not being oral surgeons, nor having any such background. After anesthetizing use dental loupes for magnification and identify the parotid papilla or the opening of the Stenson's duct on the posterior cheek adjacent to the upper second molar. Gently dilate the parotid papilla by placing the point of a conical dilator into the center or opening of the papilla and rotate the dilator back and forth for approximately 20 to 30 seconds.Next, using tweezers hold the marked end of the polyethylene tube attached to the infusate containing syringe at approximately 0.5 centimeters from the distal end and gently insert the tip of the tube into the dilated papilla. Gently advance the tube by small rotating movements to help the tubes slide, followed by readjustment of the tweezers 0.5 centimeters proximal to the previous grip. Repeat this process until the two centimeter mark reaches the parotid papilla.Next, to seal the parotid papilla and reduce spillage of the infusate back in the oral cavity, apply a cyanoacrylate on the cheek around the papilla and on the inserted tube. Once the cyanoacrylate dries, slowly inject the infusate over five minutes at a rate of 100 microliters per minute. After the infusion is complete, leave the tube in place for at least five minutes, thereby keeping the duct sealed and allowing the infusate to remain in the parotid gland.Finally, remove the tube with gentle traction and the cyanoacrylate will pull free with the tube. Fluoroscopy using a radio contrast infusion demonstrated branching of the solution through the Stenson's duct and into the parotid gland. Immuno as the chemical analysis of the parotid gland revealed positive staining for EGFP in both the ductal and acinar cells indicating successful transduction and transcription in both cell types.The main challenge in this procedure is probably introducing the polyethylene tube into the papilla, which should be done very gently and slowly.

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