|Surgical organ perfusion method for somatic gene transfer: An experimental study on gene transfer into the kidney, spleen, lung and mammary gland|
|Prev||Chapter 2. Review of the literature||Next|
Once the therapeutic gene and the target cell or tissue have been identified, the most suitable method for vector delivery has to be chosen. The most essential steps needed to obtain transgene expression efficient enough for the treatment of a disease are: 1) choice of a maximally safe and efficient vector, 2) an efficient method for delivering the vector to the target tissue, and 3) optimal control of transgene expression in the target tissue. The current gene transfer methods include both ex vivo and in vivo methods (Lyerly & DiMaio 1993). So far, the gene transfer results have usually been analyzed by using a reporter gene, such as the bacterial E. coli β -galactosidase gene (lacZ) or the firefly luciferase gene. Another way to determine transfection efficiency is to measure the mRNA of the therapeutic transgene itself or the expressed protein. Correction of the organ manifestations of the disease is the third method. Vectors may be transferred into the target cells ex vivo in a laboratory, after which the modified cells are administered to the recipient (Woolf et al. 1993, Crystal 1995b). Secondly, gene transfer can be done in vivo by delivering the vectors via various routes to the target cells of an individual (Tomita et al. 1992, Danko & Wolff 1994). The third strategy is to deliver the transgene-carrying vectors into specific cells for the transgene protein to be secreted into blood and to be transported into the target cells (Zhu et al. 1997).
Ex vivo gene transfer has been widely studied by using hepatocytes, myoblasts, fibroblasts, B cells, T cells, hematopoietic cells, vascular endothelial cells and neoplastic cells (Blaese & Anderson 1990, Medin et al. 1996). This method includes harvesting allogenic or autologous target cells from an individual, purification and culture of the cells in a laboratory, transduction of these cells with a transgene-carrying vector, usually retrovirus ex vivo, and finally, administration of the transgenic cells into the individual.
Liver cells have been obtained by liver biopsy or partial hepatectomy. The final step in gene transfer into the liver requires transplantation of the transfected cells into the host. Many different locations of transgene-producing cells have been used: intraperitoneal via a microcarrier system, gel beads placed intraperitoneally, intrasplenically through direct injection, or intrahepatically via portal infusion (Chang & Wu 1994). Grossman et al. (1994) used ex vivo retrovirus-mediated LDLreceptor gene transfer into human hepatocytes, after which the transduced hepatocytes were infused intraportally into hypercholesterolemic patients.
Kaido et al. (1996) transfected ex vivo fibroblasts with retroviral vectors carrying the hepatocyte growth factor (HGF) gene, a potent mitogen for mature hepatocytes. They found that when these transfected fibroblasts were implanted into rat spleen and the animal was intraperitoneally injected with carbon tetrachloride (CCl4), a poison for liver cells, the HGF gene transfer reduced liver injury in the treated animals compared to the controls.
Woolf et al. (1993) transfected the metanephrous tissue of mouse embryos with retrovirus-mediated β -galactosidase reporter gene ex vivo and implanted the transfected tissue under the renal capsule or cortex of adult and neonatal mice. They found that only an intrarenal site of transplantation allowed glomerular vascularization and growth, but that the transgene expression was scarce, as only 30% of the microtransplants showed any expression of the β -galactosidase gene, while the other two thirds remained negative.
The disadvantage of the ex vivo transfection method is that it is laborious, involving many phases and a need for a laboratory to culture and transfect target cells.
Ex vivo gene transfer is a technically easier method of gene transfer into organ transplants or vascular grafts before implantation, to prevent subsequent rejection or graft occlusion. Chen et al. (1994) incubated human saphenous vein and porcine jugular veins for 1, 2 and 24 hours with an AdVlacZ vector and found that the intensity of transgene expression correlated directly with the time of incubation.
In vivo gene transfer methods have the advantage of avoiding the multi-step process of ex vivo gene delivery. There is, however, a challenge in targeting the vectors efficiently enough into the appropriate target cells and tissues without affecting other organ systems. Systemic intravenous gene delivery includes vectors infused into the venous system to be transported into the target cells. This is a feasible method for treating hematological disorders or widely disseminated diseases (Baru et al. 1995). However, it yields low transfection efficiency, and organ-specific gene delivery is relatively impossible by this method. Thierry et al. (1995) used a single systemic intravenous injection of a luciferase reporter gene-plasmid DNA-liposome vector in a mouse model. They observed luciferase expression in the lung, liver and spleen four days post-injection, and the expression was slightly more efficient compared to intraperitoneal and subcutaneous administration. The expression was dependent on the DNA/liposome formulation, the DNA dose and the route of administration. The distribution of gene expression in the organs was similar to that reported by Griesenbach et al. (1998), who used a similar vector. Griesenbach´s study revealed no difference of the efficiency of gene expression in the lungs between intravenous and intratracheal administrations of the same amounts of vector.
Jaffe et al. (1992) used targeted infusion of recombinant adenovirus vectors to carry the human α1-AT gene and the β -galactosidase reporter gene into the portal vein and found that 1.1 +/- 0.2% of the hepatocytes expressed the lacZ gene. Chen et al. (1997) used portal vein infusion of the same vector to carry the human kallistatin gene into the liver in hypertensive rats and found a significant reduction of blood pressure that persisted for four weeks.
Intra-arterial infusion of vectors theoretically provides a targeted method for gene delivery into tissues. However, similarly to intravenous administration, it has been found to be quite an inefficient method for ultimate transgene expression. Schachtner et al. (1995) used intra-arterial in vivo infusion of adenoviral vectors into rat pulmonary artery via thoracotomy. They reported notable gene transfer in only 15–30% of the treated animals that survived, and the gene transfer efficiency was low. A proportion of 1–8% out of total pulmonary cells expressed the transgene. Lemarchand et al. (1994) used intra-arterial infusion of adenoviral vectors into the right upper lobe pulmonary artery of sheep, and they found transgene expression not only in the epithelial cells of airways and alveoli but also in the endothelial cells of pulmonary and bronchial vessels. Lee et al. (1998) compared intravenous and intra-arterial infusions with cationic liposome-mediated reporter gene transfer into the lungs and found that intra-arterial infusion resulted in selective transduction into the lungs, while intravenous infusion into the jugular vein resulted in disseminated transgene expression not only in lungs but also in the heart, liver and kidney. Ex vivo adenoviral infusion into the pulmonary artery and 2-hour incubation at 10°C in conjunction with lung allotransplantation resulted in weak, i.e. less than 1%, transduction of airway and alveoli epithelial cells three days after lung transplantation in a porcine model (Chapelier et al. 1996).
For renal gene transfer, Moullier et al. (1994) compared intra-arterial infusion and retrograde infusion of AdV vectors via an intrapyelic transureteral catheter. They found reporter gene expression in the proximal tubular cells of the renal cortex, but no expression in the glomeruli after intra-arterial transfection. Instead, the retrograde route of infusion resulted in transgene expression in the papillary/medullary regions, but no expression in the cortical areas. Zhu et al. (1996) used intra-arterial injection of AdV into the rat kidney with 45 minutes’ cold incubation and found transduction predominantly in the vascular cells and periglomerular as well as peritubular elements, but not in the glomeruli.
Laitinen et al. (1998) proved the safety and feasibility of intra-arterial infusion of lacZ reporter gene-carrying AdV vectors in the treatment of patients suffering from chronic leg ischemia in a phase I study. They found 0.04–5.0% transduction efficiency in arterial cells. Smooth muscle cells, endothelial cells, macrophages and tunica adventitia showed transgene expression.
Direct injection of ex vivo transduced cells into the target tissue has been used in various retroviral gene transfer projects. In addition, direct injection of vectors into tissue is a possible and simple method. Intralesional injection has been used extensively for local cancer gene transfer. However, this leads to an uneven distribution of vectors within a tumor. Cusack et al. (1996) injected recombinant lacZ gene-carrying adenoviral vectors into subcutaneously implanted human large-cell lung cancer tumors in mice. They found transduction in up to 80% of cancer cells in 1 x 1cm tumors after 24 hours. Most of the transduction was, however, found around the injection site, while only patchy focal expression was seen more peripherally. Expression was dose-dependent, the highest transduction efficiency being obtained with 1x 1010 pfu.
Successful extravascular direct injections of plasmid/liposome vectors carrying the VEGF and lacZ genes were used for gene transfer into silastic collars previously placed around rabbit carotid arteries (Laitinen et al. 1997). This led to gene transfer in mostly the adventitia and media of the artery. Efficiency was low, but the biological effects of the VEGF gene were visible in the vessel as reduced intimal thickening. Baumgartner et al. (1998), in a phase I clinical trial, administered VEGF plasmid DNA as direct intramuscular injections into the muscle of ischemic legs, and they found relief in rest pain in all the patients who presented with pain alone and alleviation of ischemic ulceration in half of the treated patients. Additionally, 80% of the treated patients demonstrated emerging collateral vessels on angiography eight weeks after treatment.
Isaka et al. (1996) used direct intramuscular injection of Sendai(HVJ) liposome solution carrying a reporter gene and a decorin, human fibrinolytic proteoglycan enzyme, gene, and they found decorin staining in muscle, kidney glomeruli, liver and lung 3 to 14 days after injection. For renal gene transfer, Lai et al. (1997) compared intraparenchymal direct injection and intra-arterial and intrarenal pelvic infusions of plasmid-liposome vectors as a gene transfer method using the lacZ reporter gene. Intra-arterial and intrarenal pelvic infusion resulted in transgene expression in the cortex and outer medulla of the kidney, but no expression was seen in the intraparenchymal injection group or in the contralateral kidney. Evidence of gene transfer was only observed in tubular epithelial cells, but not in glomerular vascular or interstitial cells, and the expression declined 3 weeks after the injection. In this study, direct intraparenchymal renal injection of vectors did not result in transgene expression.
Topical application of vectors can be used for gene transfer into skin, mucous membranes and intestinal cells. Simple topical application of vectors has not been effective. Eriksson et al. (1998) used a microseeding technique for in vivo gene transfer into skin and wounds in a porcine model. DNA plasmid solution was delivered directly into the target cells in the skin by a set of oscillating solid microneedles driven by a modified tattooing device. Some transfection was found in dermis and epidermis, and it was more efficient than transfection achieved with single injections and particle bombardment.
Adenoviral vectors were injected via laparotomy directly into the lumen of various regions of the small and large intestine by Foreman et al. (1998). They observed significant lacZ gene expression in the enterocytes lining the villi of the small intestine and in the colonic epithelial cells. Lamina propria and muscularis did not show any remarkable expression after intraluminal administration. Interestingly, by ligating the intestinal segment at both ends for two hours, to obtain an extended period of contact between the vector and the target cells during the vector administration, the transgene expression could be enhanced. Presumably, because the turnover of intestinal cells is only 2–3 days, the expression began to decrease after 3 days and was present for 8 days at the most. (Foreman et al. 1998.)
For gene transfer into the pulmonary epithelium, the application of vectors in vivo via the airway would be a feasible and minimally invasive approach. Because of the complex branching structure of the airways, it is not possible to use ex vivo gene transfer methods, and the only possible route, apart from intra-arterial delivery, is through the airways intratracheally either by instilling a transfer vector onto the epithelial surface of the airways or by aerosol delivery. Pilewski et al. (1995) reported inefficient lacZ transgene expression into epithelial cells and submucosal glands after AdV-mediated gene transfer into lung tissue xenografts of CF patients. Wilmott et al. (1996) proved the safety and feasibility of adenovirus-mediated CFTRcDNA gene transfer into the alveolar regions of lung by using intratracheal instillation of the vectors into monkeys. There were slight signs of alveolitis three days post-instillation, which resolved in a month. No remarkable long-term inflammation was found, but the expression was transient and lasted for approximately 3 weeks.
Crystal et al. (1994), in a phase I clinical trial for human CF gene therapy, used adenoviral vectors carrying the CFTR gene administered intranasally and intratracheally. The intratracheal administration was carried out via a fiberoptic bronchoscopic catheter. In bronchial brush samples, the transgene cDNA was measurable in three of the four treated patients, but quantitative assessment of the gene transfer was difficult. The authors carefully estimated that 5–14% of the airway epithelial cells were positive in one treated patient. Zuckerman et al. (1999) used intratracheal AdVCFTR cDNA administration via bronchoscope to the conducting airways of cystic fibrosis patients. This resulted in detectable but low (< 1%) gene transfer into the epithelium of the lower respiratory tract.
Katkin et al. (1995) delivered adenoviral vectors in an aerosol form to the airway pithelium of rodents. They found reporter gene expression in a maximum of approximately 10% of the large airway surface area and 25% of the surface area of the small airways. The distribution of expression was uniform throughout both lungs, whereas after intratracheal instillations, the expression is often distributed in a patchy manner. After aerosol delivery, there were no signs of inflammation, as there were after intratracheal instillations.