Name: Effective and Safe Gene Delivery to the Mouse Kidney via Slow Retrograde Renal Pelvis Injection of Adeno-Associated Virus Vectors (Video) | JoVE Journal
Uploaded: 2025-08-01T13:00:00.000+00:00
Duration: 6 min 24 s
Description: Learn a safe method for kidney gene delivery using slow retrograde renal pelvis injection of AAV vectors in mice. This technique achieves robust proximal tubule transduction without tissue damage, advancing gene therapy research.

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5 minutes

We thank Guangping Gao and James M. Wilson for providing us with the helper plasmid of AAV9 and Katja Pekrun and Mark A. Kay for the helper plasmid of AAV-KP3.

H.N. receives a royalty of AAV-related technologies licensed by Takara Bio Inc. and Capsigen Inc., serves as a consultant for biotech companies, is a co-founder of Capsigen Inc., and holds shares of Capsigen Inc. and Sphere Gene Therapeutics.

This report has detailed slow retrograde RP injection of AAV vectors as a local administration method for gene delivery to the mouse kidney. One critical step for safely performing RP injection is the adjustment of the injection rate of AAV vectors to avoid parenchymal damage to the injected kidneys. In a previous report on hydrodynamic renal pelvis injection, 100 &#181;L of plasmid DNA solution was injected into the mouse kidney within only 3 s24. This fast injection is essential for increasing t...

Effective and safe methods for gene delivery to the kidney in small and large experimental animals provide crucial opportunities to advance basic kidney research and explore innovative therapeutic approaches. Establishing such methods is particularly relevant to gene therapy for various kidney diseases. Recent advancements in clinical genetic testing have revealed a higher prevalence of monogenic renal disorders than previously estimated, affecting approximately 30% of patients with chronic kidney diseases (CKDs)1. CKD is characterized by progressive renal damage, ultimately leading to kidney failure with no truly effective disease-modifying therapies. Hemodialysis and kidney transplantation are currently available treatments; however, they have limitations that significantly impair the quality of life for patients with CKD2. Thus, gene therapy, a clinically proven powerful approach to treating genetic diseases, has garnered significant attention in recent years as a novel avenue for the effective treatment of genetic kidney diseases leading to CKD3,4,5,6. This approach can directly address the root causes of the diseases and potentially offer a cure.
Despite the steady advancements in the field, gene delivery to the kidney remains a significant challenge in both research and therapeutic settings. Efficiently targeting kidney cells with therapeutic genes is difficult due to the organ&#39;s complex structure and unique physiological barriers, including the glomerular filtration barrier, which is composed of glomerular endothelial cells, the glomerular basement membrane, and podocyte foot processes. Various strategies have been explored for kidney-targeted gene delivery, including direct renal cortex (RC) injection7,8, renal artery (RA) injection9,10,11, renal vein (RV) injection12,13,14, renal pelvis (RP) injection15,16,17,18,19,20,21,22,23,24,25 and intravenous (IV) injection19,26,27,28,29,30 of viral or non-viral gene delivery vectors, as well as ultrasound and microbubble-mediated nucleic acid delivery31,32. These approaches demonstrated variable transduction efficiencies or functional outcomes, often yielding inconsistent results that are difficult to reconcile (reviewed in references3,33,34,35). Gene delivery to the kidney is still underdeveloped, making direct comparisons between vector systems and methodologies challenging. However, localized delivery methods, which facilitate the administration of highly concentrated agents directly to the kidney, have generally achieved better results than systemic IV delivery. In addition, adeno-associated virus (AAV) vectors, with their robust safety profiles and proven track record of clinical translation36,37, have been the most studied and are regarded as the leading choice for renal gene transfer.
Common AAV serotypes and engineered capsids have demonstrated effective transduction of mesangial and mesenchymal cells in the kidney following IV injection in mice28,30,38. Notably, recent studies have shown that IV administration of AAV9 can effectively transduce podocytes in mouse models of nephrotic syndrome and Alport syndrome, although this effect is not observed in healthy kidney38,39, and that IV administration of an engineered AAV2 capsid can exclusively target glomerular endothelial cells in both healthy and inflamed kidneys in mice40. Historically, renal tubules have been challenging to transduce via the IV route. However, it has recently been discovered that RP injection of select AAV capsids, such as AAV-KP141, can achieve transduction of proximal tubules at levels more than an order of magnitude higher than those achieved with the benchmark AAV9 capsid38. Although further improvements are needed, these advancements open new avenues for basic kidney research and gene therapy, offering opportunities that were previously unattainable.
In the present article, slow retrograde RP injection of AAV vectors is detailed as an effective and safe method for gene delivery to the mouse kidney38. As a representative example of this technique, AAV-KP3 was chosen as an exemplary capsid based on the discovery that it is one of the six capsids (i.e., AAV-KP1, AAV-KP2, AAV-KP341, AAV-DJ42, AAV2G943, and AAV2.7m844) that showed enhanced renal transduction compared to AAV9 following RP injection in mice38. Notably, slow retrograde RP-injected kidney tissues showed no apparent tissue damage in contrast to those treated with hydrodynamic approaches previously reported23,24. Moreover, AAV miniprep vectors45 crudely purified by quick and simple centrifugal ultrafiltration (i.e., CU AAV vectors) achieved renal transduction levels approximately 70% of those attainable by AAV vector preparations purified by the standard cesium chloride (CsCl) gradient ultracentrifugation (i.e., CsCl AAV vectors) when administered at the same vector genome titer. Thus, the method and observations presented here provide a valuable and practical resource for researchers seeking to enhance gene delivery to the kidney in mice and potentially larger animals.

<table><tbody><tr><td>0.45 &amp;mu;m PVDF syringe filter</td><td>MilliporeSigma</td><td>SLHVR33RS</td><td></td></tr><tr><td>1.5 mL microcentrifuge tube&amp;nbsp;</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>15 mL polypropylene tube</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>16% paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>250 mL centrifuge bottle</td><td>Fisher Scientific</td><td>055641S24</td><td></td></tr><tr><td>2F Silicone tubing</td><td>Sai Infusion Technologies</td><td>SIL-2-50&amp;nbsp;</td><td></td></tr><tr><td>25G butterfly needle</td><td>Medex Supply</td><td>26708</td><td></td></tr><tr><td>2-methylbutane</td><td>MilliporeSigma</td><td>MX0760</td><td></td></tr><tr><td>2x qPCR reaction buffer</td><td>Fisher Scientific</td><td>43-676-59</td><td>Power SYBR Green PCR Master Mix</td></tr><tr><td>30G needle</td><td>Becton-Dickinson &amp;amp; Co</td><td>305106</td><td></td></tr><tr><td>50 mL polypropylene tube</td><td>Corning</td><td>430291</td><td></td></tr><tr><td>5-0 monofilament suture</td><td>Ethicon</td><td>Y463G</td><td></td></tr><tr><td>5 M NaCl</td><td>Lonza</td><td>51202</td><td></td></tr><tr><td>6-0 absorbable suture</td><td>Ethicon</td><td>J489G</td><td></td></tr><tr><td>7.5% Mini-PROTEAN TGX Precast&lt;br/&gt; Protein Gels, 10-well, 30 &amp;micro;L</td><td>Bio-Rad</td><td>4561023</td><td></td></tr><tr><td>Amersham ImageQuant 800 Western&lt;br/&gt; Blot Imaging System (Cytiva)</td><td>Cytiva</td><td>29459405</td><td></td></tr><tr><td>Amicon 100 kDa MWCO, 15 mL tube</td><td>Millipore Sigma</td><td>UFC910024</td><td></td></tr><tr><td>Anti-aquaporin 2 (AQP2) antibody</td><td>Abcam</td><td>ab199975</td><td>A marker for collecting duct cells</td></tr><tr><td>Anti-Na-K-2Cl cotransporter 2&lt;br/&gt; (NKCC2) antibody</td><td>StressMarq Biosciences</td><td>SPC-401</td><td>A marker for thick ascending limb&lt;br/&gt; cells</td></tr><tr><td>Anti-rabbit IgG Alexa Fluor 647&lt;br/&gt; antibody</td><td>Jackson ImmunoResearch</td><td>111-605-144</td><td></td></tr><tr><td>Anti-Wilms Tumor 1 (WT1) antibody</td><td>Abcam</td><td>ab89901</td><td>A marker for podocytes</td></tr><tr><td>Beckman Coulter OptiSeal Tube,&lt;br/&gt; Polypropylene, 29.9 mL</td><td>Fisher Scientific</td><td>NC9691210</td><td></td></tr><tr><td>Beckman Coulter OPTISEAL TUBES</td><td>Fisher Scientific</td><td>NC9611575</td><td></td></tr><tr><td>Benzonase</td><td>Sigma-Aldrich</td><td>1016970001</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>5470</td><td></td></tr><tr><td>Centrifugal ultrafiltration tube</td><td>MilliporeSigma</td><td>UFC9100</td><td>Amicon 100 kDa MWCO, 15 mL</td></tr><tr><td>Ceramic bead tube</td><td>Fisher Scientific</td><td>15-340-154</td><td></td></tr><tr><td>Cesium chloride</td><td>Sigma-Aldrich</td><td>C3032-1KG</td><td></td></tr><tr><td>Clamp applying forceps</td><td>Fine Science Tools</td><td>00071-14</td><td></td></tr><tr><td>Closed-loop heat pad</td><td>Stryker Medical</td><td>8002-062-012</td><td></td></tr><tr><td>Cotton-tipped applicator</td><td>Puritan</td><td>806-WC</td><td></td></tr><tr><td>Cover glass</td><td>Fisher Scientific</td><td>12-541B</td><td></td></tr><tr><td>Cryostat microtome</td><td>Tanner Scientific</td><td>TN50</td><td></td></tr><tr><td>Curved forceps</td><td>Fine Science Tools</td><td>11052-10</td><td></td></tr><tr><td>Curved-type micro vessel clip</td><td>Kleinert-Kutz</td><td>65145</td><td>Clamp for the renal artery and renal&lt;br/&gt; vein</td></tr><tr><td>Dialysis cassette</td><td>Fisher Scientific</td><td>PIA52976</td><td></td></tr><tr><td>DNA extraction kit</td><td>Qiagen</td><td>57704</td><td>QIAamp MinElute Virus Spin Kit</td></tr><tr><td>D-Sorbitol</td><td>Sigma-Aldrich</td><td>S6021-1KG</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium-&lt;br/&gt; high glucose</td><td>Corning</td><td>15-013-CV</td><td>DMEM-high glucose (4.5 g/L)</td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>15-575-020</td><td></td></tr><tr><td>Ethanol</td><td>Decon Laboratories</td><td>2716GEA</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>VWR</td><td>89510-186</td><td></td></tr><tr><td>Gas-tight glass syringe</td><td>Hamilton</td><td>1710 TLL</td><td></td></tr><tr><td>Glutaraldehyde solution</td><td>Sigma-Aldrich</td><td>G6257</td><td></td></tr><tr><td>Heat therapy pump</td><td>Kent Scientific</td><td>HTP-1500</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H4034-100G</td><td></td></tr><tr><td>Hoechst 33342</td><td>Invitrogen</td><td>H3570</td><td></td></tr><tr><td>Homogenizer</td><td>Fisher Scientific</td><td>15-340-163</td><td></td></tr><tr><td>Human embryonic kidney (HEK) 293&lt;br/&gt; cells</td><td>Agilent</td><td>240073</td><td></td></tr><tr><td>Ice bucket</td><td>Fisher Scientific</td><td>07-210-104</td><td></td></tr><tr><td>Infusion syringe pump</td><td>Harvard Apparatus</td><td>70-4507</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Critical Care</td><td>NDC 66794-017-10</td><td></td></tr><tr><td>L-glutamine</td><td>Fisher Scientific</td><td>25030-081</td><td></td></tr><tr><td>Liquid blocker pen</td><td>Ted Pella</td><td>22309</td><td></td></tr><tr><td>Lotus tetragonolobus lectin (LTL)-&lt;br/&gt; Fluorescein</td><td>Vector Laboratories</td><td>FL-1321-2</td><td>A marker for proximal tubules</td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>MilliporeSigma</td><td>M1028</td><td></td></tr><tr><td>Meloxicam</td><td>VetOne</td><td>NDC 86136-012-10</td><td>Analgesic</td></tr><tr><td>Microscope glass slide</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Mounting medium</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td>Needle holder</td><td>Fine Science Tools</td><td>12501-13</td><td></td></tr><tr><td>OCT compound</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td>Ophthalmic ointment</td><td>Bausch &amp;amp; Lomb</td><td>NDC 24208-313-34</td><td></td></tr><tr><td>PE-10 polyethylene tube</td><td>BD</td><td>427401</td><td>Inner diameter: 0.28 mm</td></tr><tr><td>Penicillin &amp;amp; Streptomycin Mix</td><td>Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Fisher Scientific</td><td>10010-049</td><td></td></tr><tr><td>Plastic cryomold</td><td>Sakura Finetek</td><td>4566</td><td></td></tr><tr><td>Poloxamer 188 Non-ionic Surfactant&lt;br/&gt; (10%)</td><td>Fisher Scientific</td><td>24-040-032</td><td></td></tr><tr><td>Polyethylene Glycol 8000</td><td>Fisher Scientific</td><td>BP233-1</td><td></td></tr><tr><td>Polyethylenimine (PEI)</td><td>Polysciences</td><td>23966</td><td></td></tr><tr><td>Povidone iodine</td><td>Ricca Chemical Company</td><td>395516</td><td></td></tr><tr><td>Power Blotter&amp;ndash;Semi-dry Transfer&lt;br/&gt; System</td><td>Fisher Scientific</td><td>PB0010</td><td></td></tr><tr><td>Proteinase K solution</td><td>Fisher Scientific</td><td>25530049</td><td></td></tr><tr><td>Rabbit anti-Anti-Adeno-Associated&lt;br/&gt; Virus (AAV), VP1, VP2, VP3</td><td>American Research Products</td><td>03-61084</td><td></td></tr><tr><td>Sarkosyl</td><td>Fisher Scientific</td><td>BP234-500</td><td></td></tr><tr><td>Screw cap 2 mL tube</td><td>Corning Axygen</td><td>SCT-200-SS-O-S</td><td></td></tr><tr><td>Silver Stain Kit</td><td>Sigma-Aldrich</td><td>PROTSIL1-1KT</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Scientific</td><td>S271</td><td></td></tr><tr><td>Sodium phosphate dibasic&lt;br/&gt; (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>MilliporeSigma</td><td>S0876</td><td></td></tr><tr><td>Sodium phosphate monobasic&lt;br/&gt; (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>MilliporeSigma</td><td>S0751</td><td></td></tr><tr><td>Sorbitol</td><td>MilliporeSigma</td><td>S6021</td><td></td></tr><tr><td>Standard pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td></td></tr><tr><td>Sterile surgical drape</td><td>SAI Infusion Technologies</td><td>PSS5-1519</td><td></td></tr><tr><td>Straight-type micro vessel clip</td><td>Fine Science Tools</td><td>00396-01</td><td>Clamp for the ureter</td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools</td><td>14058-09</td><td></td></tr><tr><td>T225 flask</td><td>Corning</td><td>431082</td><td></td></tr><tr><td>Technocut Scalpel #11</td><td>Myco Medical Supplies Inc.</td><td>6008T-11</td><td></td></tr><tr><td>Tissue forceps</td><td>Roboz Surgical Instrument</td><td>RS-5155</td><td></td></tr></tbody></table>

effective and safe gene delivery to the mouse kidney via slow retrograde renal pelvis injection of adeno-associated virus vectors

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

The development of effective in vivo tools and methods for gene delivery to the kidney is crucial for advancing basic kidney research and gene therapy for kidney diseases. In addition, growing awareness of monogenic kidney diseases, driven by advanced genetic testing, underscores the potential of gene therapy to treat and even cure difficult-to-treat genetic kidney diseases. In this regard, adeno-associated virus (AAV) vectors have garnered increasing attention as a robust platform for in vivo gene delivery; however, the most effective and safest method for AAV vector-mediated gene delivery to each therapeutically relevant cell type in the kidney has not yet been fully established. Here, the method of slow retrograde renal pelvis (RP) injection of AAV vectors is detailed, and its high potential to transduce mouse kidneys is demonstrated when used with appropriately selected AAV capsids. Moreover, this method is shown to be effective not only with the standard cesium chloride ultracentrifugation-purified AAV (CsCl AAV) vectors but also with centrifugally ultrafiltered AAV (CU AAV) vector minipreps, which can be prepared rapidly and simply using techniques that do not require specialized AAV vector expertise. As an example, this study demonstrates slow RP injection of both CsCl and CU AAV-KP3 vectors results in robust transduction of proximal tubules in the mouse kidney with no apparent tissue damage, unlike previously reported hydrodynamic approaches that inevitably lead to tissue damage. Thus, this study highlights that slow retrograde RP injection of select AAV capsid-derived vectors is an effective and safe method for gene delivery to the kidney. This method will be applicable to a broad spectrum of kidney research, including gene therapy studies. The use of CU AAV vector minipreps will significantly increase throughput and improve the time and cost efficiency needed to generate preliminary data and obtain crucial insights.

A recent study has identified a small subset of AAV capsids that exhibits remarkable enhancement of renal transduction compared to AAV9 following RP injection38. The identified subset includes AAV-KP3, a chimeric AAV capsid generated by DNA shuffling41. Therefore, AAV-KP3 was chosen for RP injection in this study, along with AAV9 as the benchmark. AAV9-CAG-tdTomato vector (AAV9) and AAV-KP3-CAG-tdTomato vector (KP3) were produced in HEK293 cells by an adenovirus-free plasmi...

This report details the localized injection method of AAV vectors via the renal pelvis for effective and safe gene transfer into the mouse kidney.

The development of effective in vivo tools and methods for gene delivery to the kidney is crucial for advancing basic kidney research and gene therapy for ...

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.

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Learn a safe method for kidney gene delivery using slow retrograde renal pelvis injection of AAV vectors in mice. This technique achieves robust proximal tubule transduction without tissue damage, advancing gene therapy research.

Effective and Safe Gene Delivery to the Mouse Kidney via Slow Retrograde Renal Pelvis Injection of Adeno-Associated Virus Vectors (Video) | JoVE Journal

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

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