Transduction of Renal Cells in Vitro and in Vivo by Adeno-Associated Virus Gene Therapy Vectors

Abstract

Abstract. There has been an increasing interest recently in the possibility of treating renal diseases using gene therapy. The ability to pursue gene therapy for renal diseases has been limited by the availability of an adequate system for gene delivery to the kidney. Adeno-associated virus (AAV) is a defective virus of the parvovirus family that has a number of properties attractive for renal gene delivery: recombinant AAV contains no viral genes; expression of genes delivered by these vectors does not activate cell-mediated immunity; the virus is able to transduce nondividing as well as dividing cells; and both wild-type and recombinant AAV integrate into the host chromosome resulting in long-term gene expression. Studies were performed to determine whether AAV can deliver reporter genes to kidney cells in vitro and in vivo. These studies show that AAV can deliver reporter genes with approximately equal efficiency to human mesangial, proximal tubule, thick ascending limb, collecting tubule, and renal cell carcinoma cells in primary culture. Immortalized mouse mesangial cells are transduced at a much greater efficiency. Transduction can be enhanced by pharmaceutical agents up to sevenfold in primary cells (transducing up to 20% of primary cells per well) and as much as 400-fold in immortalized mesangial cells. AAV delivered in vivo by intraparenchymal injection results in at least 3 mo of reporter gene expression in tubular epithelial, but not glomerular or vascular, cells at the injection site. These data indicate that AAV can deliver genes to renal cells both in vitro and in vivo resulting in prolonged gene expression, and thus AAV can be a useful tool for renal gene delivery.

Activation of Piezo1 downregulates renin in juxtaglomerular cells and contributes to blood pressure homeostasis

“…Animal care and experimental procedures were in accordance with the Guidelines for the Care and Use of Laboratory Animals (NIH Publication, 8th Edition, 2011). Recombinant AAV-mediated Piezo1-knockdown mice were generated by AAV2/9-Piezo1-shRNA virus injection, following a protocol modified from methods described previously [ 50 , 51 ]. Briefly, the kidneys of all mice were exposed and injected with 10 µL of AAV2/9-Piezo1-shRNA (1.1*10 12 vg/mL) per site for a total of 7 sites per kidney.…”

Protective Effects of PPARγ on Renal Ischemia-Reperfusion Injury by Regulating miR-21

“…Preoperative fasting was performed for 8–12 h before surgery, and the rats were anesthetized intraperitoneally by administering pentobarbital sodium (50 mg/kg). When fully anesthetized, the left kidney was exposed via a 1.5–2 cm incision along the median abdominal line, and vehicles were injected into three to four sites with recombinant AAV [ 12 ]. Four weeks after the injection, antagomiR-21 and premiR-21 (RiboBio, Guangzhou, China) were used for miR-21 regulation.…”

SIK2 protects against renal tubular injury and the progression of diabetic kidney disease

“…(AAV9) vector containing the promoter of CDH16, a tubular-specific marker [54,55], and the coding sequence of HSF1 (i.e., AAV9-pCDH16-HSF1), which is a gene therapy approach to primarily transduce cells within the renal tubules [56][57][58]. Overexpression of HSF1 in tubules was confirmed by IHC staining and immunoblot analysis (Figure 6a).…”