FIELD OF THE INVENTION The present invention is generally in the field of gene delivery and concerns conditions involving trauma-related damage in tissue or organs.

PRIOR ART 1. Zhu, N. et al., Science, 262: 209-211,1993.

2. Brigham, U. S. Patent No. 5,676,954.

3. Gorman, S. C. M., WO Publication No. 9640962 4. Boasquevisque, CH, et al., J Thorac Cardiovasc. Surg., 114 (5): 783-791 (1997).

5. Dalesandro, J., etal., J Thorac Cardiovasc. Surg., 111 (2): 416-421 (1996).

6. Ardehali, A., et al., J : Thorac Cardiovasc. Surg, 109 (4): 716-719 (1995).

7. Arancibia-Carcamo, et al., Transplantation, 65 (1): 62-67 (1998).

8. Yano, M., et al., J Thorac Cardiovasc. Surg., 114 (5): 793-801 (1997).

9. Matsumoto, T., etal., J : Vasc. Surg, 27 (1): 135-144 (1998).

The above references will be acknowledged in the text below by indicating their number (as shown in brackets) from the above list.

BACKGROUND OF THE INVENTION Various methods for in vivo gene transfer have been described wherein functional genes are carried in recombinant expression constructs capable of mediating expression of these genes in host cells. Several of these methods are based on transferring the exogenous gene by using viral vectors such as, for example, adenoviruses, retroviruses or parts thereof. Other methods for transferring exogenous genes mainly into mammalian cells, tissues or organs are achieved with the aid of liposomes comprising the exogenous DNA. The most widely used liposomes are positively charged liposomes, hereinafter"cationic liposomes" wherein the exogenous DNA is bound to the liposome rather than encapsulated within it.

Liposomes have also been used in the attempt to target the exogenous gene to a specific cell or organ.

Brigham described organ specific gene expression wherein the nucleic acid was complexed by ionic interaction to cationic liposomes. The method was especially described for transient expression of foreign genes in cells of lungs and kidneys.

Gorman, S. C. M., et al., described pharmaceutical compositions comprising a formulation of a soluble complex of a recombinant expression construct and a mixture of a neutral lipid and a cationic lipid which are used for introducing functional human genes into various tissues, especially lung tissue Liposome-mediated gene transfer to lung isografts was described wherein grafts were infused in vivo or ex vivo with liposomes comprising chloramphenicol acetyl transferase cDNA (4). Chloramphenicol acetyl transferase activity was greatest in donor lungs and hearts and minimal in livers and kidneys.

Ex vivo liposome-mediated transfection of a chloramphenicol acetyl transferase gene was further described in a rabbit model of allograft cardiac transplantation (5). Direct gene transfer into donor mouse hearts which were transplanted into recipient mice was also shown wherein a cytomegalovirus-luciferase DNA plasmid in cationic liposomes was injected

directly into the myocardium of the donor hearts resulting in expression of the cytomegalovirus-luciferase gene in the donor hearts after their transplantation In all the above-mentioned experiments, the liposomes used for transferring genes into the transplanted recipient were of the cationic type. Wherein such liposomes are used, the substance carried by the liposome is attached to the liposome. As a result, such liposomes cannot carry more than one substance, e. g. cannot comprise a DNA molecule together with a protein.

A combination of liposomes and adenovirus was also demonstrated for gene transfer to the corneal endothelial of rabbits Furthermore, in vivo and ex vivo transfection of pulmonary artery segments in lung isografts was described wherein a proximal segment of a pulmonary artery was isolated and blocked for 20 mins. and during reperfusion the arterial segments were injected ex vivo or in vivo with either adenovirus constructs or liposomes comprising the chloramphenicol acetyl transferase cDNA (8). Transgene expression was observed in proximal pulmonary artery segments after in vivo and ex vivo exposure.

Gene transfer was also shown to inhibit late graft failure of canine vein graft (9). In vivo gene transfer of endothelial cell nitric oxide synthase inhibited intimal hyperplasia of autogenous vein grafts implanted in limbs with poor distal runoffs in dogs. The transfection of the gene was carried out in a liposome prepared by reconstituting the hemagglutinating virus of Japan.

GENERAL DESCRIPTION OF THE INVENTION In accordance with the present invention, it has been found that liposomes are highly effective in delivery of active ingredients to traumatized tissue or organs and may thus be useful in treating or inhibiting progress of trauma-related damage in tissue or organs.

In accordance with the invention there is provided a method for treatment or prevention of trauma-related damage in an organ or tissue of an individual, comprising:

(a) encapsulating an active ingredient in liposomes to obtain loaded liposomes, the active ingredient being selected from the group consisting of- (i) nucleic acid molecules comprising an expressible sequence encoding a damage-preventing or a damage-reducing expression product, said sequence being under expression control of a promoter allowing expression of said expressible sequence in the target cells of said tissue or organ, (ii) a damage-preventing or damage-reducing proteinaceous substance, and (iii) a combination of (i) or (ii); and (b) parenterally administering an effective amount of the loaded liposomes to the individual.

The treatment or prevention, in accordance with the invention, may be aimed at a priori inhibiting occurrence of a damage, reducing a damage after its occurrence, as well as to inhibit the progression of the damage.

The present invention also provides a pharmaceutical composition for use in the treatment or prevention of trauma-related damage in an organ or tissue, comprising a pharmaceutical acceptable carrier, and an effective amount of liposomes loaded with said active ingredient.

The present invention further provides use of said active ingredient in the preparation of a parenteral composition for the treatment or prevention of trauma-related damage in an organ or tissue, in which said active ingredient is encapsulated in a liposome to yield loaded liposomes.

The term"effective amount"means an amount of the active ingredient which is effective in achieving the desired therapeutic effect, namely, reduction or prevention of damage to a traumatized organ or tissue. The effective amount, as will readily be understood, depends on a variety of factors including the type of damage to be treated, the severity of the damage, the age of the individual, the gender, the individual's weight, the administration regime or the administration rate (e. g. whether the active ingredient is administered once or more dailv, etc.), the type

of liposome in which the active ingredient is encapsulated, etc. In addition, the effective amount also depends on the mode of administration, namely whether the active ingredient is administered systemically, or whether the active ingredient is administered proximal to the target organ or tissue, as will be specified below. An effective amount may at times be an amount which will yield damage-preventing or damage-reducing effect, after a single administration, or may be an amount which will yield a damage-reducing or damage-preventing effect after a plurality of administrations. What constitutes to be an effective amount may easily be determined by an artisan based on routine experiment, e. g. in in vitro or in vivo.

"Trauma-related damage"encompasses a variety of situations where an organ or a tissue is subjected to stressful conditions which may affect the organ's normal function. In accordance with one embodiment of the invention, the trauma-related damage results from a transplantation procedure. Typically transplantation of an organ into a recipient elicits an immune activity of cells of the immune system of the recipient against the transplanted organ. If such an activity is not treated, it may eventually result in rejection of the transplanted organ by the recipient. In accordance with the invention, compositions comprising immune suppressing substances are perfused to the transplanted organ either prior to transplantation (by direct perfusion of the composition into the organ or by soaking the organ in the composition of the invention) or by parenteral administration, following transplantation thereof to the recipient. Such immune suppressing substances may for example, be cytokines capable of suppressing activity of immune cells, or substances which are co-stimulatory blocking agents, i. e. proteins which block the receptors on the transplanted organ which are recognized by the immune active T-cells. Examples of such co-stimulatory blocking agents are soluble CTLA4Ig and anti-CD40 ligand. Any other agents capable of reducing the immune action against the transplanted organ may also be comprised in the composition in accordance with the invention.

By an additional embodiment, the trauma-related damage results from resection. One example for such damage is resection following removal ci a

malignant tumor. In such case, the composition of the invention may comprise immune stimulating proteins such as cytokines, e. g. IL-2, Interferon-y, etc. which stimulate the immune system and reduce the chances of recurrence of the treated malignancy in the treated organ. Substances which have specific anti-cancer activity such as, for example, thymidine kinase from herpes virus, may also be used in accordance with this embodiment of the invention. In accordance with the invention, it has been shown that transfer of genes or proteins into a target organ following resection, results in efficient transfer of the gene or protein into the target organ and efficient expression of the genes or proteins in the organ.

By an additional embodiment of the invention, the trauma-related damage results from a chronic viral infection. One such example is chronic hepatitis C infection of the liver. To date, such chronic infections are typically treated by administering Interferon-a to the individual suffering from the viral infection. In accordance with the invention, it has been shown that damage resulting from chronic viral infection may be efficiently treated by the method of the invention, i. e. by administering an effective amount of liposomes comprising anti-viral agents such as, for example, Interferon-a, to the treated individual.

In accordance with the invention, trauma-related damage may also result from perfusion injuries such as Ischemia. For the first time, in accordance with the invention, it has been shown that genes administered to the target organ following Ischemia using the method of the invention, may be efficiently expressed in the organ and thus reduce or prevent Ischemia-related damage.

Although the above-mentioned embodiments are preferred embodiments in accordance with the invention, they should in no way be construed to be limiting and the method of the invention may be used for treatment or prevention of any other trauma-related damage.

Liposomes may be selected from a wide variety of liposomes known in the art. Prefered in accordance with the invention are non-cationic liposomes, namely liposomes which either bear essentially no charge (neutrally-charged liposomes) or liposomes which bear a negative charge (negatively-charged liposomes). The

liposomes used in accordance with the invention have the advantage that the ingredients which they carry are encapsulated within the liposome. This enables to include more than one such ingredient in the liposome, such as for example, a DNA construct together with a peptide.

Preferably, small sized liposomes will be used in accordance with the invention. Such liposomes will have a size in the range of about 50 nM to about 400 nM, preferably in the range of between about 100 nM to about 300 nM and most preferably in the range of about 180 nM to about 220 nM. However, larger liposomes may also be used in accordance with the invention being in the size range of from about 0.1 pm to about 5 pm, preferably from about 0.5 Sn to about 2 /2 son and most preferred from about 1 pm to about 2 pm.

The liposomes may comprise saturated and/or unsaturated phospholipids.

Examples of phospholipids are hydrogenated and non-hydrogenated soybean-dervied phospholipids, egg yolk phospholipids, dimyristoyl phosphatidyl choline (DMPC), dimyristoyl phosphatidyl glycerol (DMPG), etc. Liposomes may optionally comprise also cholesterol. The liposomes may be formulated and administered in a variety of physiologically compatible carriers, typically an aqueous solution, e. g. saline.

The manner of preparation of liposomes and their tailoring to suit a specific need is within the knowledge generally available to the artisan and elaboration on this topic is outside the scope of this writing. Non-limiting examples of methods for the preparation of liposomes is by rehydration of dehydrated lipid compositions, reverse-phase evaporation (RPE) (Kameda et al., Exp. Cell Res., 173: 56,1987), protein-cochleate technique (Gould-Fagerite, et al., Gene, 84: 429,1989) and others.

The term"target cell"will be used to denote a cell in a tissue or organ which is the target of the treatment or prevention in accordance with the invention.

Although this may generally not be required in accordance with the invention, the liposomes may comprise a variety of targeting agents anchored to the liposome membrane. An example of a targeting agent is a member of a binding

couple with the other member being a molecule which is displayed on the surface of the target cells. Examples of such binding couples are: antibody-antigen, where the antibody is anchored in the liposome membrane and the antigen being an antigen specific for the target cells; ligand-receptor couple, with the ligand being anchored in the liposome membrane and the receptor being a specific receptor displayed on the target cells; sugar-lectin couple, the lectin being anchored in the liposome membrane and the sugar being displayed on the surface of the target cells; etc.

The active ingredient may be one or both of a nucleic acid molecule or a proteinaceous substance. In the case of a nucleic acid molecule, it comprises an expressible sequence which once expressed in the target cells yields an expression product which protects the target, tissue or organ from trauma-related damages.

The term'protecting fom damages"may include direct protection, namely, protecting the cells directly from such damage, by acting within the cells, e. g. by interacting with a damaging agent to neutralize it, or, interacting with a damage-inducing biochemical pathway, etc; or may be an expression product which affects the environment of the target cells such as to avoid damage or limit the damage. An example of expression products of the former type are such which protect from chronic viral infection such as for example, Hepatisis C or apoptosis.

Examples of expression products of the latter type are a variety of cell-cycle or cell-growth regulators such as interleukin, or other cytokines which are secreted from the cell and regulate activity of other cells, e. g. lymphocytes, macrophages, etc. Specific examples of such expression products are, for example, interleukin (IL-2, IL-4, IL-10, Interferon-a, Interferon-y), and co-stimulating blocking agents such as soluble CTLA4Ig and anti-CD40 ligand which interact with cell membrane protein and block the interaction of T-cell receptors with such proteins.

The proteinaceous substance which may be used as the active ingredient, may, for example, be any of the above-mentioned expression products.

In accordance with one embodiment of the invention, the liposome includes both a nucleic acid molecule encoding an expression product, as well as the

expression product itself. In this way, both an immediate effect inflicted by the proteinaceous substance itself as well as a long-term effect resulting from continuous expression of the proteinaceous substance (the expression product) may be achieved.

The nucleic acid molecule may encode a single expression product or at times a single nucleic acid molecule may comprise more than one expressible sequence thus yielding expression of two desired expression products. In addition, a liposome may also at times comprise two or more nucleic acid molecules, each one comprising one or more expressible sequences encoding a desired expression product.

The composition of the invention may be administered by a variety of parenteral administration routes, e. g. intra-aortal, intravenous, intraperitoneal, etc. It may be administered systemically, either to a peripheral blood vessel, typically a vein, conveniently accessed by the physician. In accordance with one preferred embodiment, the administration to a blood vessel proximal to the target tissue or organ, such that the liposomes flow directly from the administration site into the target tissue or organ.

In accordance with one embodiment of the invention, the target organ or tissue is the liver. By a preferred embodiment, in order to obtain high expression of the desired DNA sequence in the liver, the composition of the invention is administered directly into the bile duct of the treated individual. Typically, this will be carried out by an operation procedure which enables direct access to the bile duct. In accordance with the invention, it has also been shown that administration of a desired DNA sequence through the bile duct in trauma conditions results in very high and specific expression of the desired DNA sequence in the liver (see examples below). Specific trauma states of the liver may include, for example, liver transplantation or partial hepatectomy in patients of liver cancer, chronic viral diseases, etc.

Although, as mentioned above, in order to obtain efficient expression of a desired DNA sequence in the liver, it is preferred to administer the composition of

the invention comprising the desired sequence through the bile duct, at times, it may also be advantageous to administer the composition of the invention via the liver portal vein.

The invention will now be described in the following non-limiting examples.

EXAMPLES Materials and Methods Expression vectors The plasmid pCI-luc was constructed by the insertion of the firefly luciferase gene (Xho I-Xba I fragment of pGL3-basic (Promega)] into Xho I and Xba I sites in pCI plasmid (Promega). The plasmid pCI-luc-PRE was constructed by the insertion of the posttranscriptional regulatory element (PRE) of hepatitis B virus (Huang. Z and Benedict Y., Mol. Cell Biol (95) 15: 3864-9; Huang, Z and Benedict. Y, J : Krol (94) 68 3193-9) into Not I and Sal I sites in the pCI-luc plasmid.

Liposome preparation A teret-butanol solution of egg phosphatidylcholine (EPC), or combination of EPC and dimyristoyl phosphatydil glycerol (DMPG) or dioleoyl phosphatydil glycerol (DOPG) or dioleoyl phosphatydil serine (DOPS) at a molar ratio of 90: 10, respectively, was prepared by dissolving the lipids in tert-butanol (Riedel De-Haen, 90% of the volume) and H20 (10% of volume) to 10% w/v solution. The mixture was mixed with plasmid DNA, dissolved in H20 to a ratio of 100: 1 (w/w) lipids to DNA and then freeze dried. The resulting dried lipids and DNA powder was reconstituted in several stages to form liposomes: First H20 was added to 30% w/v and the mixture was shaken until a homogenous solution was obtained. Then a solution of 150 mM NaCI was added in 2 stages to form 10% w/v solution of multillamelar liposomes, 0.5-1. O prn in size.

Liposome size reduction The liposome solution was first filtered throuah a 15 prn filter to elininate large aggregates from the solution. Then, the solution was filtered through 0.2 and 0.1 pm polycarbonate filters in a LipoFast Lsarge apparatus (Avestine Inc., Canada) to form liposomes of 170-200 nm.

Gene transfer after partial hepatectomy in rats: Male Wistar rats weighing 150-200 g were used. Under light diethyl ether anesthesia, 70% partial hepatectomy was performed, comprising resection of the medium and left lateral lobes according to Higgins and Anderson (Higgins, G. M. et al., =4rch Pathol. 12: 186-202,1931). 2.5 mg of pCi-luc in multilamellar liposomes (600 nm) composed of EPC, EPC-DMPG, EPC-DOPG or EPC-DOPS was injected into the mesenteric vein 1 day after partial hepatectomy. When the size of liposome was reduced, that was injected immediately after 70% partial hepatectomy. Rats were sacrificed 1 day after injection to obtain the tissue sample of several organs.

Gene transfer after total liver ischemia and reperfusion in rats : Male Wistar rats weighing 150-200 g were anesthetized by light diethyl ether. The portal vein, the hepatic artery and the choledochus were occluded"en bloc"with a vascular clamp placed hepatic hitum. Liver occlusion was maintained during 20 mins. 2.5 mg of pCl-luc in multilamellar liposomes composed of EPC-DMPG was injected into mesenteric vein immediately, 30,60,90 or 120 mins. after removing the clamp. Animals were killed 1 day after injection and the luciferase activity in several organs was measured.

Gene transfer in rat orthotopic liver transplantation model : Male Wistar rats weighing 300-350 g were used in this experiment. Both donor and recipient were anesthetized by isoflurane. Liver transplantation was performed according to the technique described by Kamada and Calne (Kamada.

N, et al., Surgery, 93: 64-69,1983). The graft was orthotopicallv transplanted by

means of suprahepatic vena cava anastomosis with a running suture (7-0 Prolene ; Ethicon) and portal vein and infrahepatic vena cava connection with a cuff technique after 1 hr of cold preservation. Hepatic artery was not reconstructed.

After completing vascular anastomoses, 2.5 mg of pCl-luc in multilamellar lipsomes composed of EPC-DMPG was injected into mesenteric vein. The bile duct was anastomosed with an intraluminal splint. The recipient was sacrificed 1 day after injection of liposomes.

Gene transfer in pig orthotopic liver transplantion model : Outbred pigs weighing 10-15 kg were premedicated with i. m. atropine sulfate (STEROP) and tiletamine and zolazepam (zolatil 100; Virbac). They were intubated and maintained anesthesia on a mixture of enflurane, nitrous oxide, and oxygen. The graft was orthotopically transplanted without a porto-systemic bypass.

The suprahepatic vena cava and portal vein were reconstructed by an end to end anastomosis, using a running suture (4-0 and 6-0 Prolene, respectively). Right before reperfusion, pCl-luc-PRE in multilamellar liposomes composed of EPC-DMPG was injected into portal vein. The infrahepatic vena cava was also reconstructed by an end to end anastomosis (5-0 Prolene) and the hepatic artery was by an end to side aorta to aorta anastomosis (6-0 Prolene). For reconstruction of the bile system, cholecystoduodenostomy (4-0 PDS II; Ethicon) was performed. At several time points after transplantation, liver tissues were taken with ultrasound- guided needle biopsy.

Luciferase assay: Tissues from rats and pigs were quickly frozen in liquid nitrogen and pulverized into a fine powder. Tissue powders were homogenized and lysed in cell culture lysis reagent (Promega). After a 30-min. incubation at room temperature, the samples were frozen and thawed three times and briefly centrifuged to remove cell debris. The luciferase activity present in 20 pl lysate was determined by the

addition of 100 pl luciferase assay reagent (Promega) with a luminometer (Biocounter M2000).

RESULTS: Example 1: Luciferase activity in rat livers 1 day after the injection of liposomes containing DNA into untreated or hepatectomized mice Male Lewis rats weighing 200-230g were divided into 2 groups. Rats of one group were anesthetized by diethyl ether and 70% partial hepatectomy was performed as described above. After 24 hours, 2.5 mg of the luciferase expression vector pCI-luc (firefly luciferase gene inserted into pCI plasmid (Promega)) encapsulated in liposomes (EPC-DMPG) were injected into the portal vein of the rats of the 2 groups. Liver biopsy was taken from the rats 2 days post injection and luciferase activity in the liver cells was measured.

As seen in Table 1 below, high luciferase activity was detected in the livers of the rats which were first hepatectomized as compared to the livers of the rats which were not hepatectomized, in which no luciferase activity was detected. The above results demonstrate the effect of trauma on the high expression of a desired DNA sequence in a target organ following gene transfer into the traumatized animal.

Table 1 Treatment before liposome Luciferase activity (pg/mg protein) injection Average + SD (n=2) 70% hepatectomy 31 5.6 Untreated 0

Example 2: Gene transfer following resection A. 70% partial hepatectomy was performed on male rats as described above.

One day after partial hepatectomy, multilamellar liposomes of different lipid compositions encapsulating the luciferase expression vector pCI-luc: (prepared as explained in"Materials and Methods"above) were injected into the mesenteric vein of the rats. One day later, the rats were sacrificed, tissue samples were obtained from their livers and luciferase activity in the liver samples was determined using the luficerase assay as described above.

As seen in Table 2 below, liposomes composed of different kinds of lipids are capable of transferring the gene into liver cells.

Table 2 Luciferase Activity in Rat Livers, 1 day following Resection and Injection of DNA Encapsulated in Liposomes of Different Types Liposome Amount of n DNA Average Type Hepatectomy Amounts Place of Luciferase (%) (mg) Injection Activity (fg/mg protein) EPC 70 2 1.5 Portal vein 606 EPC-DMPG 70 2 1.5 Portal vein 282 EPC-DOPG 70 2 1.5 Portal vein 214 EPC-DOPS 70 2 1.5 Portal vein 80.3

B. 70% partial hepatectomy was performed on rats as explained above.

One day following hepatectomy, an EPC-DMPG liposome comprising a luciferase expression vector was injected into the mesenteric vein of the rats. One day later, the rats were sacrificed and tissue was prepared from various organs of the rats.

As seen in Table 3 below, maximal luciferase activity was found in tissue obtained from liver of the above rats with some activity found also in their spleen

tissues and lung tissues. No substantive activity was found in any of the other tissue samples.

Table 3 Luciferase Activity in Several Organs, 1 day after Injection of EPC-DMPG Liposome-Encapsulated Luciferase Expression Vector in Partial (70%) Hepatectomized Rats Organ Luciferase Activity (fg/mg protein) Liver 658 + 372* spleen 161 _ 2.6 Lung 120 10.6 Kidney 13.8 13.8 Heart 0 Pancreas 0 mesenteric lymph nodes 14.5 14.5 Thymus 0 Testis 0 Brain 0 bone marrow 0 * mean SD (n =3) The above results show that injection of liposomes comprising an expression vector to the mesenteric vein of partially hepatectomized rats results in the expression of the gene comprised in the liposomes mainly in the liver of the rats.

Example 3: Gene transfer following Ischemia A. Liver occlusion was carried out on rats as described above during 20 mins.

Liposomes composed of EPC-DMPG comprising the luciferase expression vector were injected into the mesenteric vein immediately after reperfusion or at different times following reperfusion. The animals were sacrificed one day alier

injection of the liposomes. Tissue sample was obtained from their livers and luciferase activity in the tissue samples was determined as described above. As seen in Table 4 below, the highest luciferase activity was obtained when the EPC-DMPG liposomes were injected immediately following reperfusion with a sharp drop in luciferase activity when liposomes were injected 30 mins. or more after reperfusion.

Table 4 Effect of Injection Time Following Reperfusion on Luciferase Activity in the Liver, 1 day after injection of EPC-DMPG Liposome- Encapsulated Luciferase Expression Vector Injection Time DNA Place of Luciferase Following n Amounts Injection Activity Reperfusion (mg) (mean SD) (min) 0 3 2.5 Portal vein 1317 461 30 3 2.5 Portal vein 60.6 14.8 60 3 2.5 Portal vein 109 + 102 90 3 2.5 Portal vein 55.8 40.0 120 3 2.5 104+75.2 B. Tissue samples were obtained from various organs of the rats injected with the EPC-DMPG liposomes comprising the luciferase expression vector immediately after reperfusion as described in Example 4A above.

As seen in Table 5 below, the maximal luciferase activity was found in tissue obtained from the liver of these rats, with very low activity found in their spleens, lungs and hearts and no substantive activity found in any of the other tissue samples.

Table 5 Luciferase Activity in Several Organs, 1 day after Injection of EPC-DMPG Liposome-Encapsulated Luciferase Expression Vector in total Liver Ischemic (20 min) Rats Organ Luciferase Activity (fg/mg protein) mean SD (n 3) Liver 1317 + 461 * spleen 105 43.4 Lung 63. 9 + 6.6 Kidney 0 Heart 107 + 54.4 Pancreas 48. 2 + 48.2 mesentericlymph nodes 0 Thymus 0 Testis 0 Brain 0 bonemarrow 0 * Injection immediately after reperfusion Example 4: Gene transfer following transplantation Liver transplantation was performed in male rats as described above.

Following transplantation, a liposome composed of EPC-DMPG comprising the luciferase expression vector was injected into the mesenteric vein of the transplanted rats. 1 day following liposome injection, the transplanted rats were sacrificed and tissues were prepared from various parts of their organs.

Luciferase activity was determined in the obtained tissue samples in accordance with the assay described above.

As seen in Table 6 below, substantive luciferase activity was found in tissues obtained from the livers of the transplanted rats. Low luciferase activity was apparent in tissues obtained from spleen lungs and mesenteric lymph nodes of the rats and no substantive activity was found in tissues obtained from the other organs of the transplanted rats.

Table 6 Luciferase Activity in Several Organs, 1 day after Injection of EPC-DMPG Liposome-Encapsulated Luciferase Expression Vector in Liver Transplanted Rat Organ Luciferase Activity (fg/mg protein) (N = 1) Liver 1699 spleen 173 Lung 64. 7 Kidney0 Heart 0 Pancreas 35. 4 mesenteric lymph nodes 960 Thymus 0 Testis 0 Brais0 bone marrow 0 The above results show that it is possible to obtain expression of a gene comprised in a liposome mainly in the livers of rats receiving liver transplantation.

Example 5: Gene transfer by small liposomes following liver resection Rats were partially hepatectomized as described above and immediately after partial hepatectomy small liposomes (180 nm) containing the luciferase expression vector were injected into the mesenteric vein of the rats. One day following injection of the liposomes the rats were sacrificed and tissue was obtained from their livers. Luciferase activity in the liver tissue was determined using the luciferase assay described above.

As seen in Table 7 below, it is possible to obtain gene activity in rats following resection and injection of small liposomes comprising very small amounts of the expression vector comprising the expressed gene.

Table 7 Luciferase Activity comprising EPC-DMPG in Rat Livers following Resection and Injection of Small Liposomes (180 nm) containing luciferase expression vector Expression vector amount (mg) Luciferase activity (fg/mg protein) 0. 1 360 0. 5 222 1 112 1. 5 192 Example 6: Gene transfer in pig liver following transplantation Liver transplantation was carried out in outbred pigs as described above.

Immediately before reperfusion, liposomes comprised of small EPC-DMPG (180 nm) and containing the luciferase expression vector were injected into the portal vein of the pigs. Liver tissue was taken from the transplanted pigs with ultrasound-guided needle biopsy and the luciferase activity in the obtained tissue was determined using the luciferase assay as described above.

As seen in Table 8 below, substantive luciferase activity was detected in Table 8 Luciferase Activity in Pig Liver, 1 day after liver Transplantation and Injection of EPC-DMPG Liposome-Encapsulated Luciferase Expression Vector Amount of injected Liposome size (nm) Luciferase Activity DNA (m) (f/mv l) rotein) 75 180 266 The above results show that gene transfer using liposomes in transplanted animals is not restricted to rats.

Example 7: Luciferase activity in organs of rats following injection of EPC-DMPG liposome-encapsulated luciferase into the bile duct EPC-DMPG liposomes encapsulating the luciferase expression vector pCI-luc (described above) were injected into the bile duct of rats by an operation procedure. One day after injection of the lipsomes the rats were sacrificed and biopsies were taken from various of their organs. Luciferase activity was measured in the various organs as described above.

As seen in Table 9 below, injection of the liposomes into the bile duct of the rats resulted in high luciferase activity in the liver of the injected rats while no luciferase activity was detected in other organs of the injected rats. These results demonstrate the possibility of expressing a desired DNA sequence at a high level, specifically in the liver, by injection to the bile duct.

Table 9 Luciferase Activity in Several Organs of Rats, 1 day after Injection of EPC-DMPG Liposome-Encapsulated Luciferase into the Bile Duct Organ Luciferase Activity (fg/mg protein) mean + SD (n = 3) Liver 8920 + 1880 Spleen 0 Lung 0 Kidney 0 Heart 0 Pancreas 0 Mesenteric lymph nodes 0 Thymus0 Testis 0 adrenal gland 0 Brain 0

Example 8: Luciferase activity in the livers of rats, 1 day after Injection of EPC-DMPG Liposome-Encapsulated Luciferase Expression Vector into the bile duct of Untreated or Hepatectomized Rats Male Lewis rats were divided into two groups as described in Example 1 above. One group was 70% partially hepatectomized while the other group was untreated. EPC-DMPG liposomes encapsulating 0.1 mg of the pCI-luc luciferase expression vector were injected into the bile duct of the rats of both groups by an operation procedure. Liver biopsy was taken from the rats one day after injection of the liposomes and luciferase activity in the liver cells was measured as described above.

As seen in Table 10 below, luciferase activity was detected both in livers of untreated mice as well as in livers of hepatectomized rats but the extent of activity was substantively higher in the livers of the hepatectomized rats. These results demonstrate that combining trauma and injection into the bile duct results in high expression of a desired DNA sequence in the liver.

Table 10 Luficerase Activity in the liver of a rat, 1 day after Injection of EPC-DMPG Liposome-Encapsulated Luficerase Expression Vector into the Bile Duct Amount of Place of % of Size of Luciferase DNA (mg) Injection hepatectomy liposomes (nm) activity (fg/mg protein) 0.1 Bile duct 0 800 7950 0.1 Bile duct 70 800 29100

Example 9: Luciferase expression in the liver of pigs following injection of lipsome-encapsulated DNA EPC-DMPG liposomes encapsulating the luciferase expression vector were injected into the bile ducts of pigs by an operation procedure. Liver tissue was taken from the injected pigs as described above at various times following liposome injection and the luciferase activity in the liver of the injected pigs was determined as described above.

As seen in Table 11 below, luciferase activity could be detected in the livers of the injected pigs 1 to 6 days following injection of the liposomes. Thus the expression of a desired DNA sequence may be obtained in livers of pigs following injection of the sequence encapsulated in liposomes into the pigs bile duct.

Table 11 Luciferase Expression in the Liver following Injection of Liposome-Encapsulated DNA into Pigs Weight Liposome DNA Injection Luciferase (kg) size (nm) amount site (pg/mg of protein) Pig# Sex (mg) day 1 day2 day3 day6 2790 Male 10 500-800 6. 5 Bile duct 0.45 N. D. 0.14 0.10 2789 Female N. D. 0 0 2812 Male 14 500-800 15 Bile duct 0.22 N. D. 0.13 0.14 2811 Female 16 500-800 15 Bile Duct 0.13 N. D. 0.10 0

N. D.: not determined