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

Hideyuki Hakui; Anusha Sairavi; Ranjan Das; Taisuke Furusho; Hiroyuki Nakai

doi:10.3791/67716

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Method Article

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

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DOI:

10.3791/67716

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August 1st, 2025

Hideyuki Hakui¹ , Anusha Sairavi¹ , Ranjan Das¹ , Taisuke Furusho¹ , Hiroyuki Nakai¹^,²^,³

¹Department of Molecular and Medical Genetics, Oregon Health & Science University, ²Department of Molecular Microbiology and Immunology, Oregon Health & Science University, ³Division of Neuroscience, Oregon National Primate Research Center

Summary

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

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.

Introduction

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 th....