Nephron segment-specific gene expression using AAV vectors

Abstract

AAV9 vector provides efficient gene transfer in all segments of the renal nephron, with minimum expression in non-renal cells, when administered retrogradely via the ureter. It is important to restrict the transgene expression to the desired cell type within the kidney, so that the physiological endpoints represent the function of the transgene expressed in that specific cell type within kidney. We hypothesized that segment-specific gene expression within the kidney can be accomplished using the highly efficient AAV9 vectors carrying the promoters of genes that are expressed exclusively in the desired segment of the nephron in combination with administration by retrograde infusion into the kidney via the ureter. We constructed AAV vectors carrying eGFP under the control of: kidney-specific cadherin (KSPC) gene promoter for expression in the entire nephron; Na+/glucose co-transporter (SGLT2) gene promoter for expression in the S1 and S2 segments of the proximal tubule; sodium, potassium, 2 chloride co-transporter (NKCC2) gene promoter for expression in the thick ascending limb of Henle's loop (TALH); E-cadherin (ECAD) gene promoter for expression in the collecting duct (CD); and cytomegalovirus (CMV) early promoter that provides expression in most of the mammalian cells, as control. We tested the specificity of the promoter constructs in vitro for cell type-specific expression in mouse kidney cells in primary culture, followed by retrograde infusion of the AAV vectors via the ureter in the mouse. Our data show that AAV9 vector, in combination with the segment-specific promoters administered by retrograde infusion via the ureter, provides renal nephron segment-specific gene expression.

Comparison of Gene Delivery to the Kidney by Adenovirus, Adeno-Associated Virus, and Lentiviral Vectors After Intravenous and Direct Kidney Injections

“…In more recent years, several groups have used RU or SC injection techniques for gene transfer similar to the one we use in this study. 6,9,10,25 Injection through the renal artery or retrograde through the renal vein into the kidney has also had some success with various serotypes of AAV. 23,26,27 These studies did not compare different vector systems for kidney gene delivery.…”

“…We hypothesized that IV delivered vectors would suffer the filtering effects of the glomerulus and this would attenuate transduction in downstream tubule cells of the nephron. We hypothesized that delivery by the RU [5][6][7][8][9][10] route could theoretically avoid these problems, but could be limited by running upstream against the natural flow of solutes from the nephrons. Similarly, SC delivery might avoid both problems, but may instead suffer from entrapment of vectors outside of tubules and limit their ability to reach other kidney cells.…”

Lipid rafts are required for effective renal D1dopamine receptor function

“…The adeno‐associated virus (AAV) vectors harboring hD 1 R‐WT (AAV‐D 1 R‐WT) or the mutant ∆hD 1 R‐C347A (AAV‐∆hD 1 R‐C347A), with the expression controlled by a CMV promoter were constructed using the plasmid pAV‐FH (Vigene Bioscience Inc, Rockville, MD, USA) as previously described 15,17,18 …”

“…On the seventh day of siRNA infusion, blood pressure was measured under pentobarbital anesthesia using Cardiomax II to confirm the presence of elevated blood pressure. D 1 R‐WT and ΔD 1 R 347A packaged in AAV9 vectors were infused into the renal tubules via the retrograde ureteral route, as described previously 17,18 . Briefly, the distal portion of the ureter closest to the bladder and the renal artery supplying the target kidney were clamped off with micro‐venous clips in pentobarbital‐anesthetized mice.…”

Effects of Tissue Pressure on Transgene Expression Characteristics via Renal Local Administration Routes from Ureter or Renal Artery in the Rat Kidney

“…In kidney-targeting gene delivery, selection of administration route is important for determining the therapeutic target and in gene functional analysis. Additionally, local administration is desirable in order to minimize gene and nucleic acid distribution in systemic blood flow in future clinical settings [14,15]. So far, approaches from the renal artery [1,4,5,16] and vein [2,3] were mainly studied as local transfection to the kidney.…”