|Surgical organ perfusion method for somatic gene transfer: An experimental study on gene transfer into the kidney, spleen, lung and mammary gland|
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Following successful gene transfer into the glomerular cells of a kidney ex vivo, the applicability of this method for gene transfer was explored in pigs in vivo. The first two in vivo closed-circuit organ perfusions were continued for 60 minutes. Four days after the operation, β -galactosidase reporter gene expression was seen practically only in glomerular cells. Expression was seen in 9% to 58% of the glomeruli, the mean rate being 47%, as determined from 32 sections taken randomly from various sites (upper, middle and lower pole) of the kidney (Table 5). Within the glomeruli themselves, 2 to 50% of the cells were estimated to exhibit expression. No expression was observed in the cells of the other segments of the kidney, except in some vascular endothelial cells.Two other in vivo kidney perfusions lasting for 120 minutes were performed. Four days later, lacZ gene expression was observed in 23 to 75% of the glomeruli, the mean being 58% (Table 5). In individual glomeruli, expression was seen in most mesangial and endothelial cells as well as in epithelial podocytes. In certain segments, every glomerulus appeared to be positive with seemingly all cells exhibiting expression (Fig. 3). In this experiment, expression was only seen in scattered endothelial cells, while the other parts of the vascular system and the epithelial cells of the proximal and distal tubuli were negative.
Table 5. Adenoviral gene transfer into porcine kidneys using an in vivo closed-circuit perfusion method. Summary of procedures and gene transfer efficacy.
|Number of animals||Perfusion time (min)||Following time (days)||% glomeruli infected (mean)|
|2 3||60 60||4 Biopsy on day 7, 28||9–58 (47) 38–66 (48)|
|2 2||60 120||Biopsy on day 7, 84 4||some scant (d84) 23–75 (58)|
|1 (control)||60||Biobsy on day 7, 28||0|
Figure 3. . β -galactosidase expression in the kidney glomeruli four days after 60 minutes in vivo closed-circuit perfusion. a) magnification X102, b) magnification X205. Fig. 3c. The whole glomerulus and small arteriolus expressing transgene, magnification X410.
Four animals kidneys perfused for 60 minutes, including the control perfusion, were maintained postoperatively for four weeks. One kidney perfused with the viral solution showed some shrinkage after four weeks, while the other three were macroscopically normal. There was some adherence of the renal capsule to the surrounding organs due to the previous manipulation. The reason for the shrinkage of one kidney is not clear. Some lacZ gene expression was seen in the biopsies taken on day 7, but after four weeks the expression had diminished markedly. Two animals with kidneys perfused with adenoviral solution for 60 minutes were maintained for up to 12 weeks. Some lacZ gene expression was observed in the kidney biopsies one week after the perfusion, but practically no expression was seen at 12 weeks.
The spleen was perfused by placing the cannules 5 cm distal to the hilum and clamping the artery and vein just proximal and distal to the cannules, respectively. During the perfusion, there was a distinct line between the perfused and non-perfused parts on the surface of the spleen, indicating that there is apparently no anastomosing circulation between the proximal and distal parts of the porcine spleen. The proximal part of the non-perfused spleen remained dark red, while the perfused part was bright red because of the high oxygen content and the low (0.17) hematocrit value in the perfusate. When normal circulation was restored after the perfusion, the line disappeared and the distal spleen retained its dark red color. There were two unsuccessful operations, which ended in death from cardiac arrhythmia before cannulation of the splenic vessels. These experiments were excluded. Gene transfer into the spleen by closed-circuit perfusion in vivo resulted in relatively effective and evenly distributed reporter gene transduction in different types of splenic cells, as examined after four days in all of the eight successful experiments. β -galactosidase gene expression was seen in the macrophages situated around the arterioles and the arterial capillaries which terminate in the perifollicular zone between the white and the red pulp (Fig. 4) and in the red pulp (Fig. 5). Some smooth muscle cells and endothelial cells of the white pulp central arteries and arterioles leaving the white pulp also expressed the reporter gene (Fig. 6). The 60-minute perfusions led to more intense transgene expression in the spleen than the 30-minute experiments when evaluated visually.
Figure 4. β -galactosidase expression in the perifollicular area (dark arrowhead) of the spleen four days after the perfusion. Lymphoid follicle (open arrow), central artery (open arrowhead). X-gal staining, magnification a) X102, b) X205.
Figure 5. β -galactosidase expression in the red pulp of the spleen (open arrowhead), splenic trabeculae (arrow), magnification a)X102, b)X205.
Gene transfer into the lung by closed-circuit warm perfusion in vivo was performed successfully in eight farm pigs. One perfusion was continued for only 30 minutes and discontinued earlier because of cardiac arrhythmia. This animal, however, recovered from the operation normally. The other seven perfusion experiments were continued for 60 minutes. The pH values of the perfusate during the perfusion ranged within 7.40–7.43 and oxygen saturation in the perfusate was 98.5–99%. Seven days after the perfusion, lacZ transgene expression was seen mostly in the alveolar epithelial cells, type I and type II pneumocytes, and pulmonary capillary and arteriolar endothelial cells (Fig. 7 and 8). Some expression was also seen in bronchial and bronchiolar epithelial cells and alveolar macrophages (Fig. 9). Approximately 2–5% of the epithelial cells expressed the transgene. Immunohistochemistry revealed β -galactosidase protein in both the alveolar epithelial cells (type I and II pneumocytes) and the alveolar endothelial cells (Fig. 10).
Figure 7. β -galactosidase expression seven days after 60 minutes closed-circuit lung perfusion in the alveolar epithelial cells and macrophages a) type I pneumocyte (arrow), magnification X205 b) type II pneumocyte (arrow), magnification X410.
Figure 8. β -galactosidase expression in the wall of the small pulmonary arteriole, magnification X410.
Figure 9. β -galactosidase expression in the bronchial epithelial cells shown by arrows, magnification a) X102, b) X205.
Figure 10. Immunohistochemical staining of β -galactosidase protein in pulmonary alveolar epithelial cells (arrow) and endothelial cells (arrowhead), AEC, magnification a)X205, b)X410.
The distribution of transgene expression in the lung was relatively even throughout the specimens, except after the 30-minute perfusion, which was almost negative. This 30-minute perfusion experiment was excluded, because of the short duration of perfusion. The quantification of transgene expression was estimated by counting every nuclear-dominant blue area per section in twenty sections per experiment (Fig. 11). In places where the blue area consisted of numerous cells, it was counted as one uniformly stained single spot. When all the seven successful pulmonary gene transfer experiments with 60-minute perfusion were included, the mean count of positive exogenous gene-expressing spots was 100 transgene-expressing spots per 100 mm2 section. The count varied from 3 to 365 spots per section. The detailed results of each experiment are shown in table 6.
Figure 11. The estimated quantitity of expression in the pulmonary tissue by counting the clearly nuclear-dominant blue spots, here total of 17 spots included, magnification X102.
Table 6. β -galactosidase gene expression in the porcine lung after closed-circuit organ perfusion.
|Perfusion time/minutes||Mean count of the transgene expressing areas/100 mm2||Range of variation|
In two experiments, the untreated left control lung showed some low, presumably endogenous transgene expression in the alveolar macrophages (data not shown), but no expression was seen in the other cells of the control lung tissue.
Gene transfer trials with organ perfusion for 60 minutes using retroviral vectors for human growth hormone gene transfer into mammary gland were made on three female goats. Transduction efficiency was evaluated by measuring the growth hormone titers in the goat´s milk daily for one week after the perfusion and thereafter until the expression disappears. Some hGH gene transfer was detectable from the first day, but the expression was low in two experiments. One experiment did not result in marked expression. In the two successful experiments the titers declined markedly from the third day after the perfusion and disappeared by one month in the first trial. However, hGH expression could be measured for as long as for five months in the second trial (Fig. 12 and 13).
Figure 12. Human growth hormone (hGH) titers in the milk after 60 minutes retroviral closed-circuit perfusion of the mammary gland in the first experiment on goat.
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