EP0556197A1

EP0556197A1 - Prolonging expression of polynucleotides introduced into a cell

Info

Publication number: EP0556197A1
Application number: EP91917379A
Authority: EP
Inventors: George Y. Wu; Catherine H. Wu
Original assignee: University of Connecticut
Current assignee: University of Connecticut
Priority date: 1990-09-25
Filing date: 1991-09-23
Publication date: 1993-08-25
Also published as: JPH06503714A; EP0556197A4; CA2092319A1; WO1992005250A1; AU8628291A

Abstract

L'expression de polynucléotides introduits dans une cellule au moyen d'un complexe ciblé constitué du polynucléotide lié à un agent de liaison spécifique à la cellule peut être prolongée en stimulant la réplication de la cellule.The expression of polynucleotides introduced into a cell by means of a targeted complex consisting of the polynucleotide linked to a cell-specific binding agent can be prolonged by stimulating replication of the cell.

Description

PROLONGING EXPRESSION OF POLYNUCLEOTIDES INTRODUCED INTO A CELL

Background

A foreign gene can be targeted to, and expressed, in a cell in vivo. This can be accomplished using a soluble complex of DNA and a carrier system consisting of two linked components: 1) a polycation, e.g., poly-L-lysine, that can bind a polynucleotide in a strong but non-damaging interaction and 2) a ligand which can be targeted specifically to a cell surface molecule unique to the cell. See Wu, G.Y. and Wu, C.H. (1988) J. Biol. Chem. 2£ :14621-14624. The complex specifically binds the targeted cell and is internalized by it, resulting in the incorporation of the DNA.

The expression of exogenous DNA introduced into a cell by this method can be transient. Although short-lived expression may be desirable for some applications of gene therapy, in many other applications, prolonged expression of the poly nucleotide would be required. There exists a need for a method of introducing DNA into cells in vivo so that expression of the DNA can be made to persist. Summarv of the Invention

This invention pertains to a method of introducing a polynucleotide into a target cell and causing expression of the introduced polynucleotide to persist in the cell. According to the method, the polynucleotide is administered in vivo as a targeted polynucleotide complex which is specifically incorporated into the cell and the target cell is stimulated to replicate. Replication of the cell causes expression of the introduced polynucleotide to persist, as compared to the transient expression which can occur in the absence of stimulated replication.

The targeted polynucleotide complex comprises the polynucleotide releasably (under intracellular conditions) complexed to a cell-specific binding agent which specifically binds a molecule on the surface of the target cell. The polynucleotide can be bound to the cell-specific binding agent through a polynucleotide-binding agent, such as a polycation, which is linked to the cell-specific binding agent. The binding agent for the cell surface component can be a ligand for a cellular surface receptor, preferably a cellular surface receptor which mediates internalization of ligands by endocytosis, or it can be a receptor, such as an antibody, for a cellular surface antigen. The targeted polynucleotide complex is preferably soluble in physiological fluids.

The target cell can be stimulated to replicate by surgical or pharmacological means. For example, partial excision of regenerative organ or tissue which the cell constitutes can be performed to stimulate replication. Alternatively, a drug which stimulates cellular replication can be used.

The method of this invention is useful in gene therapy and in other applications where prolonged expression of a polynucleotide introduced into a cell is desired.

Brief Description of the Figures

Figure 1 shows the time course of targeted gene expression. Groups of two rats were injected with asialoorosomucoid (AsOR)-poly-L-lysine-polynucleotide (CAT gene) complex and at daily intervals animals were killed, liver samples removed, homogenized and equal amounts of homogenate protein assayed for CAT activity.

Figure 2 shows the effect of partial hepatectomy on targeted CAT gene expression. Rats were injected with AsOR-poly-L-lysine-polynucleotide complex. A 66% partial hepatectomy was performed 30 minutes later, and at various time points livers were assayed for CAT activity.

Figure 3 shows a Southern blot demonstrating the state of targeted DNA in livers 11 weeks after partial hepatectomy. DNA was extracted from livers, digested with restriction enzymes, and applied on an agarose gel electrophoresis together with palb-CAT standards as described below in the Exemplification. The samples were subsequently transferred to nitrocellulose and detected by hybridization to a 32_P_;_{L a}]_Deι_ea _{AT cDNA} robe . Detailed Description of the Invention

Selective incorporation of a polynucleotide into a target cell in vivo is brought about by administer¬ ing the polynucleotide in the form of a targeted complex. The targeted polynucleotide complex comprises polynucleotide linked releasably to a cell-specific binding agent which binds a surface component of the targeted cell. The polynucleotide complex is administered in vivo where it selectively binds to and is internalized by the cell.

The polynucleotide is generally DNA. Typically it comprises a structural gene encoding the product to be expressed in the target cell and appropriate genetic regulatory elements (promoter, enhancer etc.) to regulate expression of the structural gene product in the target cell. These can be carried by a vector such as plasmid or a transposable genetic element.

The preferred system for introducing the polynucleotide into the cell is described in U.S. Patent Application Serial No. 504,064 filed April 2, 1990, the teachings of which are incorporated by reference herein.

The polynucleotide complex can be made by binding the polynucleotide directly to the ligand. Preferably, the polynucleotide is bound to the ligand through a polynucleotide-binding agent. In general, the polynucleotide-binding agent is covalently bonded to the ligand. The polynucleotide-binding agent must be capable of complexing the polynucleotide so that the polynucleotide is released from the complex in a functional form after internalization within the cell. The bond should be extracellularly stable, i.e., of strength sufficient to prevent uncoupling of the polynucleotide extracellularly prior to cell internalization but it should be cleavable under appropriate conditions within the cell so the polynucleotide can be released intracellularly. For example, the bond between the polynucleotide-binding component and the polynucleotide can be a non- covalent bond based on electrostatic attraction. Preferred polynucleotide-binding agents are polycations that bind to negatively charged polynucleotides. Positively charged polycations provide secure, tight complexing in a noncovalent manner to form soluble, targetable polynucleotide complexes. The bound polynucleotide is undamaged. Suitable polycations are polylysine, polyarginine, polyornithine, basic proteins such as histones, avidin, protamines and the like.

Other non-covalent bonds that could be used consistent with linkage strategy include hydrogen bonding, hydrophobic bonding, electrost-cic bonding alone or in combination/as in: anti-polynucleotide antibodies bound to polynucleotide, and strepavidin or avidin binding to polynucleotide containing biotinylated nucleotides.

The polynucleotide complex can contain more than one polynucleotide molecule. Preferably the ratio of polynucleotide to ligand-polynucleotide binding complex is from about 0.5 to about 1. The number may vary, depending upon factors such as the effect on solubility or capillary permeability of the complex. The cell-specific binding agent can be a ligand which binds to a surface receptor of the target cell or the binding agent can be a receptor or receptor¬ like molecule, such as an antibody, which binds a ligand (antigen) on the cell surface. Preferably, the binding agent is a ligand for a cellular surface receptor which mediates internalization of the ligand by, for example, the process of endocytosis. The receptor-specific ligand can be a protein having functional groups that are exposed sufficiently to be recognized by the cell receptors. The receptor- specific ligand can also be a component of a biological organism such as a virus, or cells (bacterial, protozoan or mammalian) or artificial carriers such as liposomes.

Particular ligands will vary with the particular target cell. Glycoproteins having certain exposed terminal carbohydrate groups can be used although other ligands such as polypeptide hormones, also may be employed. For specific targeting to hepatocytes (liver cells), asialoglycoprotein (galactose- terminal) ligands are preferred. Examples of asialoglycoproteins include asialoorosomucoid, asialofetuin and desialylated vesicular stomatitis virus. These can be formed by chemical or enzymatic desialylation of those glycoproteins that possess terminal sialic acid and penultimate galactose residues. Alternatively, hepatocyte-targetable asialoglycoprotein ligands may be created by coupling lactose or other galactose terminal carbohydrates (e.g., arabinogalactan) to non-galactose-bearing proteins by reductive lactosamination. Because a variety of different receptors exist on the surfaces of mammalian cells, cell-specific targeting to other (non-hepatic) cells can involve ligands such as mannose for macrophages (lymphoma) mannose-6-phosphate glycoproteins for fibroblasts (fibrosarcoma) , intrinsic factor - vitamin B12 for enterocytes and insulin for fat cells.

In preferred embodiments, the polynucleotide complex is soluble in physiological fluids. Soluble DNA complexes can be prepared with proteinaceous ligands and polycations as polynucleotide-binding agents. The polynucleotide complex is generally administered parenterally in a physiologically- acceptable vehicle, generally in an amount sufficient to saturate receptors of the target cell in vivo.

The target cell can be stimulated to replicate either before or after (generally within 12 hours) administration of the polynucleotide.

The target cell can be stimulated to replicate by surgical or pharmacological means. In some cases, partial excision of a regenerative organ or tissue which are comprised of the cell can be performed to stimulate replication (regeneration). For example, partial hepatectomy can be performed to stimulate replication of liver cells. Pharmacological agents which stimulate replication of the target cells such as nafenopin, galactosamine and carbon tetrachloride, or analogues thereof, may be used. Factors or hormones that stimulate replication may also be used. For example, insulin and glucagon in combination can stimulate hepatocyte replication. Additional doses of the stimulant may be administered at various intervals after the initial administration (with or without the administration additional targeted polynucleotide complex) to prolong further the expression of the polynucleotide.

Unlike transient foreign gene expression which generally peaks between 1-2 days and ceases after about 4 days, the expression achieved by the method of this invention can peak at approximately 8 weeks after administration of the polynucleotide. Furthermore, expression can persist at significant levels for at least 4 months. An unusual feature of the persistent expression is the increase in the level of expression over time.

This method of prolonging gene expression has value for gene therapy of inherited disorders of metabolism in man and animals. The prolongation of the foreign gene expression makes practical the periodic, but not too frequent, administration of a gene. Further, replication of the targeted DNA increases the mass of the desired gene in the host. Therefore, stimulation of replication of recipient cells may obviate the need for readministration of the gene.

The invention is illustrated further by the following examples.

EXAMPLES

Example 1. Persistent gene expression brought about by partial hepatectomy.

Materials and Methods

Preparation of a Targetable polynucleotide Carrier - To form a carrier system capable to being targeted specifically to hepatocytes, orosomucoid was isolated from pooled human serum (American Red Cross, Farmington, CT) (Whitehead, D.H., and Sammons, H.G. (1966) Biochim. Biophvs. Acta 124.:209-211) and desialylated with insolubilized neuraminidase (Type X-A, Sigma) to form asialoorosomucoid (AsOR)

(Kawasaki, T., and Ashwell, G. (1977) J. Biol. Chem. 252:6536-6543) . Residual sialic acid was determined to be less than 10% (Warren, L. (1959) J. Biol. Chem. 2~A:1917-1975) . Poly-L-lysine, mean M_r = 3,800 (Sigma), was coupled to AsOR in a 2:1 molar ratio using l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Pierce Chemical Co.) adapted from the method of Halloran and Parker (Halloran, M.J., and Parker, C.W. (1966) J. Immunol. 96:373-378). purified, and analyzed as described previously (Wu, G.Y. and Wu, C.H. (1987) J. Biol. Chem. 262:4429-4432).

Plasmid Preparation - The palb-CAT construct was prepared by replacement of an SV40 early promoter by mouse albumin promoter (-330 to +10 base pairs) and enhancer sequences (-12 to -8.5 kilobases) (Pinkert, C.A., Ornitz, D.M., Brinster, R.L., and Palmiter, R.D. (1987) Genes & Dev. 1:268-276) in the plasmid MTBV.JT. The plasmid was cloned in Escherichia coli. isolated, and purified (Birnboim, H.C., and Doly, J. (1979) Nucleic Acids Res. 7:1513-1518). Purity was confirmed by 1% agarose gel electrophoresis demonstrating the absence of bacterial cellular polynucleotide. For hybridization studies, the CAT insert was labeled with ³²P by nick translation (Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular Cloning; A Laboratory Manual, pp. 150-161, Cold Spring Harbor Laboratory, Cold Spring Harber, NY) . Formation of a Targetable Carrier-polynucleotide Complex - The optimal proportions for complex forma¬ tion between the palb-CAT plasmid and the AsOR-poly- L-lysine conjugate were determined using an agarose gel retardation system as described previously (Wu, G.Y., and Wu, C.H. (1988) Biochemistry 27:887-892). A conjugate to DNA molar ratio of 25:1 (based on AsOR content of the AsOR-poly-L-lysine conjugate) was found to completely retard DNA migration in the gel and form soluble complexes, and this ratio was used in all subsequent experiments. All complexes were filtered through 0.45 μm membranes (Millipore) prior to injection to ensure that samples used did not contain precipitates. Complexes were found to be stable in saline or in rat serum at 37°C for at least 1 hour, and in saline at 4°C for at least 2 weeks. Targeted Gene Expression - To assess targeted gene expression, female Sprague-Dawley rats (220-250 g) in groups of two were injected intravenously with 1 ml of saline containing 580 μg of palb-CAT DNA in the form of AsOR-poly-L-lysine-DNA complex or controls, and at daily intervals animals were killed and liver samples were removed and homogenized. Homogenates were assayed for protein content (Bradford, M.M. (1976) Anal. Biochem. 72:248-254) and equal amounts of homogenate protein assayed for CAT activity (Gorman, C ., Moffat, L.F., and Howard, B.H. (1982) Mol. Cell. Biol. 2:1044-1051).

To examine the effect of partial hepatectomy on the time course of targeted gene expression, groups of rats were injected intravenously with AsOR-poly- L-lysine-DNA complex, and 30 minutes later 66% partial hepatectomies were performed (Wayforth, H.B. (1980) Experimental and Surgical Technigues in the Rat. Academic Press, New York). Animals were maintained over an 8 week period during which they were periodically killed, livers removed and homogenized, and hepatic CAT activity determined. At the 8-week point, a lobectomy was performed and animals killed at 11 weeks. All CAT assays were performed in duplicate.

State of the Targeted polynucleotide Associated with Persistent Gene Expression - To determine the state of the DNA targeted by our delivery system, DNA was extracted from liver homogenates by a phenol/ chloroform method (Rowe, D.W., Moen, R.C., Davidson, J.N., Byers, P.H., Bornstein, P., and Palmiter, R.D. (1987) Biochemistry 17:1581-1590). Samples of DNA were digested with Bstell (2 units/μg polynucleotide) which does not cut the palb-CAT plasmid, Xbal (2 units/μg polynucleotide) which cuts he plasmid at a single site, and BamHI (2 units/μg polynucleotide) which excises the CAT insert from the plasmid. All digestions were carried out at 37°C for 18 hours after which time samples were applied in increasing concentrations on an 1% agarose gel for electrophor- esis along with standard palb-CAT plasmid. The DNA was then transferred to nitrocellulose and CAT sequences detected by hybridization with a ³²P-labeled CAT cDNA probe (Southern, E.M. (1975) J. Mol. Biol. :503-517). Resuits

Targeted foreign gene expression as a function of time is shown in Figure 1, a representative assay for CAT gene expression. CAT activity was 10 units/g liver at 24 hours and 7.6 units/g 48 hours after injection. However, the expression was transient as activity declined to 4.6 units/g at 72 hours, and by 96 hours CAT activity was no longer detectable.

The effect of 66% partial hepatectomy on the time course of hepatic targeted gene expression is shown in Figure 2. Lane 3 shows that CAT activity was not detectable 24 hours after hepatectomy but was restored by 48 hours to a level of 2.4 units/g liver. This activity remained detectable well beyond the 96-hour limit seen in the transient studies shown previously. CAT activity actually increased to a maximum of 14.6 units/g liver by the 8th week and persisted at high levels, 11.3 units/g, through the 11th week post-hepatectomy. To determine the state of the targeted DNA in livers with persistent CAT gene expression, DNA was extracted from portions of DNA complex-treated livers 11 weeks post-partial hepatectomy. In Figure 3 lanes 1-3 contain palb-CAT plasmid, 0.01, 0.05, and 0.1 μg, respectively, linearized by digestion with Xbal which cuts the plasmid at a single site. Lane 4 shows the electrophoretic position of the CAT insert excised from the standard palb-CAT plasmid by BamHI. Lane 5 shows that cellular DNA from livers treated with the targetable DNA complex and analyzed 11 weeks after partial hepatectomy contained high molecular weight sequences that hybridized with the CAT cDNA probe. Digestion of this cellular DNA with BamHI, shown in lane 6, resulted in complete release of the CAT insert which migrated in a manner identical to the insert excised from standard palb-CAT plasmid (lane 4). Lanes 7-9 show that Xbal digestion of cellular DNA from livers treated with complex and analyzed 11 weeks post-partial hepatectomy resulted in the formation of some hybridizable fragments of lower molecular weight than the intact linear plasmid, but the majority of the hybridizable sequences remained present as DNA greater in size than the linear form of the plasmid. Restriction of cellular DNA by an enzyme that does not cut the plasmid, Bstell, shown in lanes 10-12, resulted in the formation of hybridizable fragments that were all greater in size than the palb-CAT plasmid. Doubling the ratios of restriction enzymes to cellular DNA as well as the duration of digestion with Xbal and Bstell did not change the restriction patterns (data not shown) . The copy number in samples of livers 11 weeks post- transfection was calculated to be approximately 18:1. Finally, lane 13 shows that cellular DNA from control livers (saline-treated) analyzed 11 weeks after partial hepatectomy demonstrated no hybrid¬ izable CAT sequences, indicating that the observed hybridization by complex-treated liver DNA was not due to nonspecific binding of the probe to any endogenous host sequences. Example 2. Persistence of gene expression brought about by pharmacological means.

A. In order to determine whether persistence of targeted gene expression could be achieved by non- surgical means, the plasmid, p9-12 albCAT containing the CAT gene driven by mouse albumin regulatory elements was complexed to our targetable DNA carrier system.

The hypolipidemic drug, nafenopin [2-methyl-2-p-(1,2,3,4-tetrahydro-1-naρhthy)- phenoxypropionic acid] , was chosen for study because of previous reports demonstrating that nafenopin is a potent stimulator of hepatocyte replication without causing hepatocellular damage. Our first objective was to determine the optimal time in which nafenopin should be administered in relation to injection of the DNA complex. To determine this, groups of rats, 200-250 gm, were pre-injected with nafenopin, 200mg/kg i.p., 12, 18 and 24 hours prior to the injection of complex or saline control. Because our previous studies had demonstrated that targeted foreign gene expression is transient, lasting a maximum of 4 days, we undertook to examine foreign gene expression after nafenopin injection, 7 davs after administration of the DNA, a time when transient expression should be absent. Rats treated with complex and nafenopin, or controls were sacrificed at the 1 week point and liver CAT enzyme activity determined as described originally by Gorman, et. ELI. , supra. Our studies demonstrated that the optimal time for pre-injection of nafenopin was 20-24 hours prior to administration of the DNA complex. Using this injection schedule, CAT gene expression was 2.3 units/mg of liver by 2 weeks and remained detectable through 9 weeks.

In order to determine the effect of repeated doses of nafenopin and replication of hepatocytes, groups of rats were pre-injected with nafenopin followed by complex as described above and subsequently injected with a second dose of nafenopin at the same dose 1 week later. After an additional week, CAT activity was assayed and found to have risen to a level of 9.3 units/mg.

In order to determine the effect of repeated doses of both complex and nafenopin, groups of rats received 2 doses each of complex plus nafenopin, 1 week apart. When CAT enzyme activity was assayed two weeks after the initial injection, CAT levels rose to 78 units/mg, a 14-fold increase over the second injection of nafenopin alone and a 34-fold increase over a single injection of complex and nafenopin. Control animals that received complex alone or nafenopin alone had no detectable CAT activity at 1 or 2 weeks after injection.

In order to assess possible hepatocellular damage, serum alanine amino transferase (ALT) levels were assayed at each time point that hepatic CAT activity was measured. The serum ALT levels remained normal throughout the course and for a single or double doses of nafenopin and/or nafenopin plus complex. A southern blot of livers obtained at the time of CAT assay demonstrated that bands corresponding to material hybridizable with a ³²P-cDNA probe to CAT sequences were present at 1, 2 and 9 weeks after injection of complex and nafenopin. Restriction of the total cellular DNA obtained from livers treated with complex and nafenopin demonstrated that the CAT gene could be excised intact from the total cellular 5 DNA indicating that the CAT gene, as expected, was intact.

We conclude that targeted foreign gene expression can be made to persist and increase by stimulation of hepatocyte replication through 0 administration of a non-toxic pharmacological agent.

B. Persistence of targeted foreign gene expression achieved by chemical partial hepatectomy.

Regeneration of the liver by hepatocyte replication can be achieved by chemical damage to 5 hepatocytes. This has been demonstrated in a variety of liver specific toxins of which galactosamine is a classic example.

In order to determine whether administration of galactosamine could be used to achieve persistence of 0 targeted gene expression, the p9-12 albCAT plasmid was again complexed with our DNA carrier system. In order to determine the optimal time for pre-injection of galactosamine, 900mg/kg galacto¬ samine was injected i.p. into groups of rats, 25 200-240gm at 16, 20, 24 and 39 hours prior to i.v. injection of complex or saline control. One week later animals were sacrificed and CAT enzyme activity determined in liver. From these data, the optimal time or pre-incubation of galactosamine was 30 determined to be 24 hours. In order to determine the time course of targeted foreign gene expression after administration of galactosamine, groups of animals were pre-injected with galactosamine 24 hours prior to complex and at varying time intervals, hepatic CAT activity was determined. In addition, serum ALT values were determined at each time point. CAT activity in liver was found to reach peak levels of 6.7 units/mg at 2 weeks and persist at least through 4 weeks after complex and galactosamine administration. Serum ALT values demonstrated levels of approximately 1,200 IU, 24 hours after injection of galactosamine indicating that mild hepatocellular injury had occurred. These data indicated that a selective hepatotoxin can be administered in a controlled manner to result in mild hepatocellular injury and hepatocyte replication with persistence of targeted gene expression.

A plasmid, p9-12albCAT, containing the CAT gene driven by mouse albumin regulatory elements, was injected iv as a targetable complex into rats. Hepatic CAT activity was transient, undetectable by 96 hrs. after injection of complex. Nafenopin, 200 mg/kg pre-injected ip 20-24 hrs. prior to the complex resulted in persistence of CAT gene expression through 2 weeks (2.3 U/mg) . When the dose of nafenopin alone was repeated 1 week after the first, CAT activity rose to 9.3 U/mg, 2 weeks after the initial injection. Furthermore, a repeated dose of both complex plus nafenopin 1 week after the first injection increased CAT 3vels 14-fold to 78 U/mg when assayed 2 weeks aft r the initial injection. Targetable polynucleotide complex alone, or nafenopin alone given at the same two time points resulted in undetectable CAT activity at 2 weeks. A time course showed that all animals that received targetable polynucleotide complex plus nafenopin produced CAT levels that remained high through at least 9 weeks. Targeted foreign gene expression can be made to increase and persist by stimulation of hepato¬ cyte replication by administration of a non-toxic, pharmacological agent.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experi¬ mentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

1. A method of introducing polynucleotide into a cell of an organism for persistent expression, comprising administering to the organism a polynucleotide complex to specifically incorporate the polynucleotide into the cell, the polynucleotide complex comprising the polynucleotide releasably linked to a cell- specific binding agent which binds a surface molecule of the cell and is internalized by the cell; and stimulating the cell to replicate.

2. A method of claim 1, wherein the polynucleotide is DNA.

3. A method of claim 1, wherein the polynucleotide comprises a gene.

4. A method of claim 1, wherein the organism is a human.

5. A method of claim 1, wherein the polynucleotide complex further comprises a polynucleotide- binding agent through which the polynucleotide is linked to the cell-specific binding agent.

6. A method of claim 1, wherein the polynucleotide- binding component is a polycation.

7. A method of claim 6, wherein the polycation is polylysine.

8. A method of claim 1, wherein the cell-specific binding agent binds a surface receptor of the cell which mediates endocytosis.

9. A method of claim 8, wherein the cell-specific binding agent is a ligand for the asialoglyco¬ protein receptor of hepatocytes.

10. A method of claim 9, wherein the ligand is an asialoglycoprotein.

11. A method of claim 1, wherein the cell is stimulated to replicate by partial excision of the organ or tissue which the cell comprises.

12. A method of claim 1, wherein the cell is stimulated to replicate by a pharmacological agent.

13. A method of claim 12, wherein the pharmaco¬ logical agent is nafenopin or an analogue thereof.

14. A method of claim 12, wherein the pharmaco¬ logical agent is galactosamine.

15. A method of introducing DNA into hepatocytes of an organism for persistent expression therein, comprising administering to the organism a DNA complex to specifically incorporate the DNA into hepatocytes, the complex comprising DNA bound to a conjugate of a ligand for an asialoglycopro¬ tein receptor and a polycation capable of complexing the DNA and releasing it within the hepatocyte; and stimulating the hepatocytes to replicate.

16. A method of claim 15, wherein the ligand for the asialoglycoprotein receptor is an asialoglyco¬ protein.

17. A method of claim 15, wherein the polycation is polylysine.

18. A method of claim 15, wherein the hepatocytes are stimulated to replicate by partial hepatectomy.

19. A method of claim 15, wherein the hepatocytes are stimulated to replicate by pharmacological means.

20. A method of claim 19, wherein the pharmaco¬ logical agent is nafenopin or an analogue thereof.

21. A method of claim 19, wherein the pharmaco¬ logical agent is galactosamine or an analogue thereof.

22. Use of a polynucleotide complex comprising the polynucleotide releasably linked to a cell- specific binding agent, for the manufacture of a medicament for introducing the polynucleotide into a cell of an organism for persistent expression of the polynucleotide under cellular stimulation by, for example, a pharmacological agent, e.g. nafenopin or an analogue thereof, or galactosamine, or an analogue thereof.

° 23. Nafenopin, or analogue thereof, for the manu¬ facture of a medicament for stimulating a cell of an organism (e.g. hepatocytes) containing a therapeutic polynucleotide (e.g. DNA) to replicate for persistent expression of the 5 polynucleotide.

24. Galactosamine, or an analogue thereof, for the manufacture of a medicament for stimulating a cell of an organism (e.g. hepatocytes) contain¬ ing a therapeutic polynucleotide (e.g. DNA) to 0 replicate for persistent expression of the polynucleotide.

25. Use of a DNA complex comprising the DNA bound to a conjugate of a ligand for an asialoglyco¬ protein receptor and a polycation, for the 5 manufacture of a medicament for introducing the DNA into hepatocytes of an organism for persistent expression of the DNA under stimulation of the hepatocytes by, for example, a pharmacological agent, e.g. nafenopin or an

30 analogue thereof, or galactosamine, or an analogue thereof.