GTMB 7 - Gene Therapy & Molecular Biology

Gene Therapy and Molecular Biology Vol 7, page 225could be used effectively to transfer naked plasmid DNAinto the kidney. The mechanism by which ultrasoundmicrobubbles enhance transgene expression in all celltypes within the kidney may largely be attributed toultrasound-mediated microbubble cavitation. It is possiblethat the cavitation may largely increase the permeability ofcapillary and tubular basement membranes, which allowsthe locally released DNA to cross through these basementmembranes and enter cells, including glomerular,interstitial, and tubular epithelial cells.The ultrasound-Optison-mediated m2Smad7/Tet-onplasmid transfer resulted in transgene expression in morethan 90% of glomerular, tubular, and interstitial cells.Gene transfer of inducible Smad7 using the ultrasoundmicrobubblesystem inhibited renal fibrosis in the ratunilateral ureteral obstruction model. The ultrasoundtreatment did not cause any abnormal histologic orfunctional changes, as evidenced by normal urinaryprotein excretion, normal glomerular and tubulointerstitialmorphology, and the lack of cellular and interstitial edemaand of local inflammation.E. Naked plasmid DNAIt has not been possible to express transgenes in thekidney by injecting naked plasmid DNA via the renalartery (Lai et al, 1997, Boletta et al, 1997). Lai et al (1997)and Boletta et al (1997) performed intrarenal arterialinjection of naked plasmid DNA while the renal arterialblood flow was interrupted but the renal venous bloodflow was not. The hydrodynamics-based transfectionmechanism did not work in either of these studies.F. AVE-type HVJ-liposome ex vivoThe AVE-type HVJ-liposome method effectivelyinduced the heat shock protein (HSP) 70 or the bcl-2 genein kidney grafts. This was effective even when the HVJliposomevector was mixed with a cold-preservationsolution (Ringer’s lactated or University of Wisconsinsolution) and infused into the renal artery just prior tostorage of the kidney at 4°C for 24 to 48 h in the samepreservation solution (Kita et al, 2003). The induction ofthe HSP70 or bcl-2 gene reduced the occurrence ofprimary non-function of grafted rat kidneys after longtermpreservation. The transgene expression in the kidneywas limited to the tubules. In contrast, Tsujie et al (2001a),as described above, observed expression exclusively in theglomeruli when they introduced plasmid DNA using theAVE-type HVJ-liposome by renal arterial injection. Thediscrepancy between the two studies may be due to thedifference in plasmid vectors used, or for other, as yetunknown, reasons.III. Kidney-targeted gene transfer viarenal veinTable 2 gives an overview of studies that have usedkidney-targeted naked plasmid DNA transfer via the renalvein. Kidney-targeted gene transfer via the renal vein canbe achieved exclusively by hydrodynamics-basedtransfectionA. Hydrodynamics-based transfectionMaruyama et al, (2002a) have developed a techniquefor transferring naked plasmid DNA into the kidney ofnormal rats by hydrodynamics-based transfection byretrograde injection of the DNA into the renal vein. Whenthis technique was performed using a lacZ expressionplasmid as the reporter gene, lacZ expression was detectedexclusively in the interstitial fibroblasts near theperitubular capillaries (PTC) of the injected kidney, asassessed by immunoelectron microscopic analysis. Nonephrotoxicity attributable to gene transfer was apparenteither by histological or functional examinations of theinjected kidney. These authors also used this method totransfect rat kidney with erythropoietin (Epo) gene.Maximal Epo expression was obtained when the vector,pCAGGS-Epo, was injected in Ringer’s solution within 5sec, and with a volume of 1.0 ml. The transgene-derivedEpo mRNA was detected by RT-PCR only in the targetedkidneys and without aberrant expression in nontargetorgans. After an injection of 100 µg of pCAGGS-Epo, theserum Epo levels peaked at 208.3 ± 71.8 mU/ml at week 5,and gradually decreased to 116.2 ± 38.7 mU/ml at week24. Transgene-derived Epo secretion resulted in significanterythropoiesis. The preparation of naked plasmid DNA issimple, compared with the preparation involved for othernonviral techniques. Moreover, the most long-term stablegene expression in the kidney has been obtained using thistechnique.The mechanism underlying the transfer of the nakedplasmid DNA into fibroblasts is unclear. The negativelycharged (Dworkin et al, 2000) PTC could be refractory tothe transfer of negatively charged naked plasmid DNAinto the endothelium.However, the PTC wall consists of an extremely thinendothelium, and fifty percent of the PTC endothelium isfenestrated, and thus highly permeable to water and smallsolutes (Lemley and Kriz, 1994). No incubation time isrequired for this technique; therefore, hydrostatic pressure(Liu et al, 1999) may be the mechanism underlying thetransfer of the naked plasmid DNA. Efficient expressionmay depend on the elevated intravascular hydrostaticpressure caused by the rapid injection of a sufficientvolume of fluid, which leads to the transfer of the nakedplasmid DNA through the PTC endothelium, despite thenegative charge. The sharp vascular resistance gradientbetween the efferent arteriole and the PTC (Lemley andKriz, 1994) probably plays a major role in the blockade ofthe retrograde stream of injected naked plasmid DNAsolution and in the dilation of the PTC, which are the mostexpandable sites in this route, resulting in transgeneexpression in the fibroblasts. Moreover, these cells mayhave the ability to take up the naked plasmid DNA.Recently, Shimizu et al, (2003) demonstrated that thetransfer of a kidney-targeted naked plasmid encoding 7ND(anti-monocyte chemoattractant protein-1) into the kidneyinterstitial cells using the technique of retrograde injectioninto the renal vein (Maruyama et al 2002a) attenuates the225