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Surgical organ perfusion method for somatic gene transfer

Surgical organ perfusion method for somatic gene transfer

An experimental study on gene transfer into the kidney, spleen, lung and mammary gland

Teija Parpala-Spårman

Department of Surgery


The progress in recombinant DNA technology has made possible the introduction of exogenous genetic material into cells. Gene therapy aims to correct a defective gene, introduce a therapeutic exogenous gene or a counteracting gene into somatic cells without modification of the germ-cell line. The most important technical interests in the field of gene therapy research have pertained to the development of safe and effective vectors and suitable methods for the delivery of the exogenous gene carrying vectors into the target cells. The aim of this study was to evaluate surgical methods used for gene delivery and to develop an effective gene transfer method for organ-specific gene transfer, primarily into the renal glomeruli.

There are genetic and acquired diseases that are candidates for gene therapy. Alport syndrome is an X-chromosome-linked disease caused by a mutation in the type IV collagen 5 chain gene, which causes a defect of the glomerular basement membrane in the kidney, leading to progressive renal failure in males. This manifestation could theoretically be prevented by the transfer of a normal 5 chain gene into the renal glomerular cells. Cystic fibrosis and 1-antitrypsin deficiency are examples of pulmonary diseases and genetic lysosomal storage diseases that are candidates for splenic gene transfer. The gene transfer strategies used so far have proved relatively ineffective. Recombinant adenovirus, retrovirus, adeno-associated virus and liposomes have been previously used as vectors. Direct injection, intra-arterial, intravenous and intratracheal delivery of vectors have been the most extensively studied methods.

This preclinical experimental work for marker gene transfer into the kidney, spleen, lung and mammary gland was done by using rabbits, pigs and goats as test animals. The adenoviral vector carrying a -galactosidase reporter gene was first infused in the renal artery of rabbits and pigs in vivo with or without pharmacological agents. This did not result in any remarkable gene transfer into the kidney. Next, the incubation time between the vector and the target cells was prolonged by ex vivo perfusion of explanted kidneys for 12 hours. Perfusion at room temperature did not improve gene transfer. When the perfusion temperature was raised to 37˚C, improved and mostly glomerular gene transfer was observed, with up to 80% of the glomeruli showing -galactosidase expression in four ex vivo experiments.

A closed-circuit organ perfusion method for in vivo gene transfer was developed in this study. The surgical perfusion experiment was tested successfully in ten in vivo perfusions of the kidney, eight of the spleen and eight of the lung in a porcine model. This method led to effective, up to 75% gene transfer into the renal glomeruli as assessed after four days. In the spleen, the perfusion method resulted in relatively effective gene transfer into perifollicular splenic cells, mostly macrophages and endothelial cells. Lung perfusion yielded transgene expression in alveolar epithelial cells, bronchiolar epithelial cells and, to a lesser extent, arteriolar endothelial cells and alveolar macrophages. Perfusion of the goats mammary gland using a retroviral vector in three experiments resulted in growth hormone secretion into the milk.

The gene transfer operation was well tolerated by the animals, and no clinical signs of inflammation were observed. No remarkable humoral immunological response against adenovirus or -galactosidase was elicited in the kidney experiments, but histological signs of inflammation as mononuclear cell clusters in the kidney and lung were seen four and seven days after the experiments. The spleen showed no macroscopic or microscopic pathologic alterations after the perfusion.


To my family

Table of Contents
List of original papers
1. Introduction
2. Review of the literature
2.1. History of gene therapy research
2.2. Applications of somatic gene therapy
2.2.1. Kidney diseases
2.2.2. Pulmonary diseases
2.2.3. Malignant diseases
2.2.4. Metabolic disorders
2.2.5. Cardiovascular diseases
2.2.6. Hematological diseases
2.2.7. Gastrointestinal diseases
2.2.8. Diseases of the nervous system
2.3. Gene transfer vectors
2.3.1. Retrovirus
2.3.2. Adenovirus
2.3.3. Adeno-associated virus
2.3.4. Other viral vectors
2.3.5. Non-viral vectors
2.4. Development of vector delivery methods
2.4.1. Ex vivo gene transfer and implantation of transduced cells or tissue
2.4.2. Intravenous gene delivery
2.4.3. Intra-arterial gene delivery
2.4.4. Direct injection of vectors
2.4.5. Topical application of vectors
2.4.6. Intratracheal gene delivery
2.5. Problems related to the gene transfer methods
2.6. Attempts to enhance transgene expression in gene transfer
3. Aims of the present study
4. Materials and methods
4.1. Adenoviral vector
4.2. Retroviral vector
4.3. Animals
4.4. Surgical procedures
4.5. Intra-arterial infusion of vectors in vivo
4.6. Ex vivo gene transfer into kidney by the closed-circuit organ perfusion method
4.7. In vivo gene transfer with a surgical closed-circuit organ perfusion method
4.7.1. In vivo gene transfer into kidney glomeruli
4.7.2. In vivo gene transfer into the spleen
4.7.3. In vivo gene transfer into the lung
4.7.4. In vivo gene transfer into the mammary gland
4.8. Histochemical analysis
4.9. Immunohistochemistry
5. Results
5.1. Gene expression after intra-arterial infusion in vivo
5.2. Gene transfer by closed-circuit perfusion ex vivo
5.3. Gene transfer by closed-circuit organ perfusion in vivo
5.3.1. Gene transfer into the kidney
5.3.2. Gene transfer into the spleen
5.3.3. Gene transfer into the lung
5.3.4. Gene transfer into mammary gland
5.4. Tissue and systemic effects of gene transfer by the closed-circuit organ perfusion method
6. Discussion
7. Conclusions
List of Tables
1. Gene therapy methods for treatment of cancer.
2. Characteristics of the most commonly used gene transfer vectors.
3. Summary of animal experiments used in this study.
4. Experimental intra-arterial infusions of virusparticles into renal artery in conjunction with various vasodilative agents.
5. Adenoviral gene transfer into porcine kidneys using an in vivo closed-circuit perfusion method. Summary of procedures and gene transfer efficacy.
6. β -galactosidase gene expression in the porcine lung after closed-circuit organ perfusion.
List of Figures
1. Schematic presentation of the closed-circuit perfusion system attached to the kidney.
2. Effective β -galactosidase expression in the kidney glomeruli after 12 hours ex vivo closed-circuit perfusion at 37˚C. Cryosection, X-gal, magnification X102.
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.
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.
5. β -galactosidase expression in the red pulp of the spleen (open arrowhead), splenic trabeculae (arrow), magnification a)X102, b)X205.
6. Weak β -galactosidase expression in the smooth muscle cells and endothelial cells of one white pulp central artery (open arrowhead) and strong expression in the arterioles leaving the white pulp (arrows) in the spleen, magnification a)X102, b)X410.
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.
8. β -galactosidase expression in the wall of the small pulmonary arteriole, magnification X410.
9. β -galactosidase expression in the bronchial epithelial cells shown by arrows, magnification a) X102, b) X205.
10. Immunohistochemical staining of β -galactosidase protein in pulmonary alveolar epithelial cells (arrow) and endothelial cells (arrowhead), AEC, magnification a)X205, b)X410.
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.
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.
13. hGH expression in the goat´s milk after retroviral perfusion of the mammary gland for 60 minutes in the second experiment.
14. Normal excretory urogram four weeks after the perfusion experiment on the left kidney (arrow).
15. Cut surface of the kidney a) four weeks after the perfusion experiment, left kidney b) non-perfused right kidney.
16. a) A normal appearing glomerulus four weeks after perfusion. PAS-HE staining, magnification X205. b) inflammatory changes in the kidney, a mononuclear cell cluster (arrow) around renal blood vessel four weeks after the kidney perfusion experiment. PAS-HE staining, magnification X315.