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

Abstract

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.