Effective and Safe Gene Delivery to the Mouse Kidney via Slow Retrograde Renal Pelvis Injection of Adeno-Associated Virus Vectors

Our goal is to develop effective ways to deliver genes to kidney cells using adeno-associated virus vectors. This will advance basic kidney research and help develop novel gene therapies for kidney diseases. A major challenge is the lack of effective methods to deliver genes to key kidney cell types, such as renal tubules and podocytes due to the organ's structural complexity, limiting progress in kidney targeted adeno-associated virus gene therapy.

We have shown that effective gene delivery to kidney cells, including renal tubules and podocytes can be achieved by selecting the right adeno-associated virus capsids and administration routes based on disease context. Dilute the adeno-associated virus vector solution using 5%Sorbitol PBS. Place the diluted solution on ice.

With a needle holder, bend and break off only the needle tip from a 30-gauge needle. Under a stereo microscope, connect the 30-gauge needle tip, four centimeters of PE10 tubing pre-attached to 15 centimeters of silicone tubing, another 30-gauge needle, and a gastight glass syringe in order from the tip. Set the connected gastight glass syringe onto an infusion syringe pump.

Using the infusion syringe pump, aspirate 90 microliters of the vector solution into the gastight glass syringe. Set the infusion mode on the syringe pump to a target volume of 50 microliters and a flow rate of 50 microliters per minute. Next, place an anesthetized mouse in a lateral position on a closed loop heat pad connected to a heat therapy pump maintained at 37 degrees Celsius.

Cover the mouse with a sterile surgical drape. Using a sterile scalpel, make a one-centimeter skin incision at the costovertebral angle level near the left kidney. Cut the underlying muscle layer along the same length as the skin incision.

Then gently press the abdomen with fingers avoiding direct contact with the kidney to expose the kidney through the incision site. Carefully remove minimal surrounding fat until the renal pelvis appears as a small white area. Use a straight type micro vessel clip to clamp the ureter first.

Then clamp the renal artery and vein together with a curve-type micro-vessel clip. Use curved forceps to insert the 30-gauge needle about three millimeters into the pelvic cavity. Start the injection of 50 microliters of vector solution using the infusion syringe pump over one minute at a flow rate of 50 microliters per minute.

Keep the needle and clamps in place for five minutes to enhance kidney exposure to the vector. Then remove both the ureter and vessel clamps. Slowly withdraw the needle while pressing a cotton-tipped applicator over the injection site to apply pressure.

Gently reposition the kidney back into the peritoneal cavity. Then suture the muscle layer with six-zero absorbable suture and then the skin with five-zero monofilament suture. Finally, inject one milliliter of saline subcutaneously into the mouse's back for fluid support.

The concentrations of vector genomes in crude lysates were relatively low, on the order of 10 to the power of 10 vector genomes per milliliter for both AAV9 and KP3 preparations, while the final centrifugally-ultrafiltered, purified AAV9 and KP3 preparations showed high vector genome titers of 3.2 into 10 to the power of 13 and 8.8 into 10 to the power of 12 vector genomes per milliliter respectively. SDS-PAGE analysis with silver staining of cesium-chloride-purified AAV9 and KP3 revealed three distinct bands for VP1, VP2, and VP3 with minimal background, indicating high purity. While centrifugally-ultrafiltered purified samples loaded at high amounts displayed protein smearing, but at lower loads, VP bands became distinct.

Two weeks after renal pelvis injection, the centrifugally-ultrafiltered KP3 vector transduced renal proximal tubules effectively, while the centrifugally-ultrafiltered AAV9 vector showed limited tubular transduction and was mostly confined to glomerular mesangial cells. Quantitative PCR revealed a 73 fold higher vector genome copy number in centrifugally ultrafiltered KP3-injected kidneys. The KP3 vector also transduced collecting duct cells as shown by colocalization with Aquaporin-2 staining while AAV9 primarily targeted thick ascending limb cells costained with NKCC2.

Off-target liver transduction was noticeably reduced with KP3 compared to AAV9. No apparent histological damage in kidneys injected with the KP3 vector was observed. Our slow renal pelvis injection method increases local adeno-associated virus vector concentration, thereby allowing efficient transduction of various kidney cell types that are otherwise difficult to target by systemic delivery.

By utilizing both the systemic and renal pelvis injection routes, we aim to identify optimal adeno-associated virus capsids and administration routes for effective and selective transduction in target kidney cell types.