AU686614B2 - Targeted delivery of genes encoding antisense polyribonucleotides - Google Patents

Description

WO 94/23050 PCT/US94/03643 -1- TARGETED DELIVERY OF GENES ENCODING ANTISENSE POLYRIBONUCLEOTIDES Background of the Invention Antisense polynucleotides are a means of specifically inhibiting unwanted gene expression in cells. They can be used to hybridize to and inhibit the function of an RNA, typically a messenger RNA, by physically blocking the binding of ribosomes or other proteins, thus preventing translation of the mRNA. Antisense polynucleotides also include RNAs with catalytic activity (ribozymes), which can selectively bind to complementary sequences on a target RNA and physically destroy the target by mediating a cleavage reaction.

Antisense polynucleotides can be in the form of small, chemically synthesized DNA or RNA oligonucleo- tides, or can be larger RNAs, such as mRNAs, biosynthetically generated in vitr or in vivo by transcription of an antisense gene. Since hundreds of copies of RNA can be synthesized from each copy of a gene, a few molecules of an antisense gene in a cell would achieve the same effect as the introduction into the cell of a large number of antisense oligonucleo- tides. The 50 to 300 times greater size of a typical mRNA allows an antisense mRNA to bind with greater affinity and specificity compared to an oligonucleotide. In addition, unlike oligonucleotides, a plasmid-bome gene can incorporate a wide variety of supplemental DNA sequences to enhance or modulate the expression of the antisense polyribonucleotide. For example, inclusion of origin sequences which direct episomal replication of a plasmid and promoter and/or enhancer sequences that sustain a high level of expression over long periods would allow long term production of an antisense polyribonucleotide. This strategy would be particularly suited for inhibiting the constitutive expression of a cellular gene or for treating chronic conditions or diseases. To achieve a similar effect with an antisense oligonucleotide would most likely require continuous or repeated intravenous administration or highly stable antisense constructs.

Summary of the Invention This invention pertains to a soluble molecular complex for targeting a gene encoding an RNA transcript to a specific cell in vivo or in vitro and obtaining production of the RNA within the targeted cell. The molecular complex comprises an expressible gene encoding a desired polyribonucleotide complexed to a carrier which is a conjugate of a cell-specific binding agent and a gene-binding agent. In antisense applications, the RNA transcribed from the delivered gene can be used to hybridize to and inhibit the function of an RNA contained within the cell. The target RNA is typically a messenger RNA. The RNA transcribed from the delivered gene can also be an RNA with catalytic activity (a ribozyme), which can selectively destroy the target RNA. The target for antisense or ribozyme-mediated inhibition i i -I i i L- U I"II can be a gene or genes of cellular origin a cellular oncogene) or of noncellular origin a viral oncogene or the genes of an infecting pathogen such as a virus or a parasite such as malaria, trypanosome, lysteria, or mycoplasma).

The cell-specific binding agent is specific for a cellular surface structure, typically a receptor, which mediates internalization of bound ligands by endocytosis, such as the asialoglycoprotein receptor of hepatocytes. The cell-specific binding agent can be a natural or synthetic ligand (for example, a protein, polypeptide, glycoprotein, etc.) or it can be an antibody, or an analogue thereof, which specifically binds a cellular surface structure which then mediates internalization of the bound complex. The gene-binding component of the conjugate is a compound such as a polycation which stably complexes the gene under extracellular conditions and releases the gene under intracellular conditions so that it can function within the cell.

The complex of the gene and the carrier is stable and soluble in physiological fluids. It can be administered in vivo where it is selectively taken up by the target cell via the surface-structure-mediated endocytotic pathway. The incorporated gene expressed and the gene-encoded product accumulates within the transfected cell.

The soluble molecular complex of this invention can be used to specifically transfect cells in vivo or in vitro to provide for synthesis of a desired product. This selective transfection is useful for antisense gene therapy and other applications which require selective genetic alteration of cells to inhibit the expression of cellular or foreign V. genes. The RNA transcript produced from the delivered gene hybridizes with its complementary RNA, inhibiting its function either by steric hindrance, or by physical S; cleavage, thereby blocking expression of the target gene or genes.

According to a first aspect the present invention consists in a soluble molecular 25 complex for targeting a gene encoding an antisense RNA to a specific cell, the complex comprising an expressible gene encoding an RNA which hybridizes to and inhibits the function of a cellular RNA, and a carrier comprising a cell specific binding agent and a gene-binding agent.

According to a second aspect the present invention consists in a therapeutic composition comprising a solution of the molecular complex of the first aspect in a physiologically acceptable vehicle.

18282.00 Doc/mtm

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2a- According to a third aspect the present invention consists in a soluble molecular complex for targeting a gene encoding an antisense RNA to a hepatocyte, the complex comprising an expressible gene encoding an antisense RNA which hybridizes to an RNA transcript produced in the hepatocyte, and a carrier comprising a ligand for the asialoglycoprotein receptor and a polycation.

According to a fourth aspect the present invention consists in a therapeutic composition comprising a solution of the molecular complex according to the third aspect, in a physiologically acceptable vehicle.

According to a fifth aspect the present invention consists in a method of blocking translation of an RNA transcript in a cell of an organism, comprising administering to an organism a soluble molecular complex comprising an expressible gene encoding an antisense RNA which hybridizes to and inhibits the function of cellular RNA, and a carrier comprising a cell-specific binding agent and a gene binding agent.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic depiction of the construction of a plasmid encoding an antisense RNA directed against hepatitis B surface antigen (pJ300.8HTD1) and a plasmid encoding an antisense RNA directed against the hepatitis core antigen gene as well as the DRI and polyadenylation site.

Figure 2 shows the reduction of HBsAg mediated by 21-mer antisense oligodeoxynucleotide and antisense mRNA-generating plasmid pJ3Q0.8HTD1 (Anti-C).

DETAILED DESCRIPTION OF THE INVENTION 0 A soluble, targetable molecular complex is used to selectively deliver a gene encoding a polyribonucleotide to a target cell or tissue in vivo or in vitro. The molecular *0*000 complex comprises the gene to be delivered complexed to a carrier made up of a binding 25 agent specific for the target cell and a gene-binding agent specific for the target cell and a gene-binding agent. The complex is selectively taken up by the target cell and the polyribonucloetide is produced there.

18282-00.DOC/mtm pr~lb WO 94/23050 PCT/US94/03643 -3- The gene, generally in the form of DNA, encodes the desired polyribonucleotide.

Typically, the gene comprises a sequence encoding the polyribonucleotide in a form suitable for transcription and post-transcriptional processing by the target cell. For example, the gene is linked to appropriate genetic regulatory elements required for transcription of the gene by a cellular RNA polymerase and processing of the primary RNA transcript by cellular proteins into a stable form of RNA, such as mRNA. These include promoter and enhancer elements operable in the target cell, as well as other elements such as polyadenylation signals and splicing signals, which determine the internal and 3'-end structure of the RNA. The gene can be contained in an expression vector such as a plasmid or a transposable genetic element along with the genetic regulatory elements necessary for transcription of the gene.

The carrier component of the complex is a conjugate of a cell-specific binding agent and a gene-binding agent. The cell-specific binding agent specifically binds a cellular surface structure which mediates its internalization by, for example, the process of endocytosis. The surface structure can be a protein, polypeptide, carbohydrate, lipid or combination thereof. It is typically a surface receptor which mediates endocytosis of a ligand. Thus, the binding agent can be a natural or synthetic ligand which binds the receptor. The ligand can be a protein, polypeptide, glycoprotein or glycopeptide which has functional groups that are exposed sufficiently to be recognized by the cell surface structure. It can also be a component of a biological organism such as a virus, cells mammalian, bacterial, protozoan) or artificial carriers such as liposomes.

The binding agent can also be an antibody, or an analogue of an antibody such as a single chain antibody, which binds the cell surface structure.

Ligands useful in forming the carrier will vary according to the particular cell to be targeted. For targeting hepatocytes, glycoproteins having exposed terminal carbohydrate groups such as asialoglycoprotein (galactose-terminal) can be used, although other ligands such as polypeptide hormones may also be employed. Examples of asialoglycoproteins include asialoorosomucoid, asialofetuin and desialylated vesicular stomatitis virus. Such ligands can be formed by chemical or enzymatic desialylation of glycoproteins that possess terminal sialic p rd and penultimate galactose residues. Alternatively, asialoglycoprotein ligands can be formed by coupling galactose terminal carbohydrates such as lactose or arabinogalactan to non-galactose bearing proteins by reductive lactosamination.

For targeting the molecular complex to other cell surface receptors, other types of ligands can be used, such as mannose for macrophages (lymphoma), mannose-6-phosphate glycoproteins for fibroblasts (fibrosarcoma), intrinsic factor-vitamin B 12 or bile acids for enterocytes and insulin for fat cells. Alternatively, the cell-specific binding agent can be a receptor or receptor-like molecule, such as an antibody which binds a ligand antigen) on the cell surface. Such antibodies can be produced by standard procedures.

The gene-binding agent complexes the gene to be delivered. Complexation with the gene must be sufficiently stable in vivo to prevent significant uncoupling of the gene 4n--~aFs~sa~la~ WO 94/23050 PCTIUJS9403643 -4extracellularly prior to internalization by the target cell. However, the complex is cleavable under appropriate conditions within the cell so that the gene is released in functional form.

For example, the complex can be labile in the acidic and enzyme rich environment of lysosomes. A noncovalent bond based on electrostatic attraction between the gene-binding agent and the expressible gene provides extracellular stability and is releasable under intracellular conditions.

Preferred gene-binding agents are polycations that bind negatively charged polynucleotides. These positively charged materials can bind noncovalently with the gene to form a soluble, targetable molecular complex which is stable extracellularly but releasable intracellularly. Suitable polycations are polylysine, polyarginine, polyornithine, basic proteins such as histones, avidin, protamines and the like. A preferred polycation is polylysine ranging from 3,800 to 60,000 daltons). Other noncovalent bonds that can be used to releasably link the expressible gene include hydrogen bonding, hydrophobic bonding, electrostatic bonding alone or in combination such as, anti-polynucleotide antibodies bound to polynucleotide, and strepavidir, or avidin binding to polynucleotide containing biotinylated nucleotides.

The carrier can be formed by chemically linking the cell-specific binding agent and the gene-binding agent. The linkage is typically covalent. A preferred linkage is a peptide bond. This can be formed with a water soluble carbodiimide as described by Jung, G. §t aL (1981) Biochem. Bioshys. Res. Commun. 101::599-606. An alternative linkage is a disulfide bond.

The linkage reaction can be optimized for the particular cell-specific binding agent and gene-binding agent used to form the carrier. Reaction conditions can be designed to maximize linkage fonnation but to minimize the formation of aggregates of the carrier components. The optimal ratio of cell-specific binding agent to gene-binding agent can be determined empirically. When polycations are used, the molar ratio of the components will vary with the size of the polycation and the size of the gene- binding agent. In general, this ratio ranges from about 10:1 to 1:1, preferably about 5:1. Uncoupled components and aggregates can be separated from the carrier by molecular sieve or ion exchange chromatography Aquapore T M cation exchange, Rainin).

In one embodiment, asialoorosomucoid-polylysine conjugate is formed with the crosslinking agent 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide. After dialysis, the conjugate is separated from unconjugated components by preparative acid-urea polyacrylamide gel electrophoresis (pH The conjugate can be further purified on the carboxymethyl functionalized column (Waters AP-1 column). See U.S. Patent Application Serial No. 08/043,008, filed on April 5, 1992, the teachings of which are incorporated by reference herein.

The gene encoding the antisense construct can be complexed to the carrier by a stepwise dialysis procedure. In a preferred method, for use with carriers made of polycations 4 k~ ~IBli(sli~WPIJOI WO 94/23050 PCTIS9403643 such as polylysine, the dialysis procedure begins with a 2M NaC1 dialyzate and ends with a NaCI solution. The gradually decreasing NaCI concentration results in binding of the gene to the carrier. In some instances, particularly when concentrations of the gene and carrier are low, dialysis may not be necessary; the gene and carrier are simply mixed and incubated.

The molecular complex can contain more than one copy of the same gene or one or more different ger Preferably, the weight ratio of gene to the carrier is from about 1:5 to 5:1, preferably about 1:2 (approximate molar ratio 1:100 to 1:200). The appropriate ratio for a particular polynucleotide and carrier can be determined by the gel retardation assay described in U.S. Patent No. 5,166,320.

The molecular complex of this invention can be administered parenterally.

Preferably, it is injected intravenously. The complex is administered in solution in a physiologically acceptable vehicle.

Cells can be transfected in vivo for transient production of the polyribonucleotide.

For prolonged production, the gene can be administered repeatedly. Alternatively, the transfected target cell can be stimulated to replicate by surgical or pharmacological means to prolong the activity of the incorporated gene. See, for example, U.S. Patent Application Serial No. 588,013, filed September 25, 1990, the teachings of which are incorporated by reference herein. Drugs that disrupt translocation or fusion of endosomes to lysosomes such as colchicine or taxol can be used to prolong expression. See U.S. Patent Application Serial No. 950,789, filed September 24, 1992, the teachings of which are incorporated by reference herein.

Delivery of the gene can be enhanced by coupling the carrier to a virus such as adenovirus. See U.S. Patent Applicaton Serial No. 950,453, filed September 24, 1992 the teachings of whirh are incorporated by reference herein.

The method of this invention can be used to selectively deliver a gene to a target cell in vivo for antisense gene therapy or other applications which require inhibition of the expression of specific cellular or foreign genes. The RNA transcript produced from the delivered gene hybridizes with its complementary RNA, inhibiting its function either by steric hindrance, or by physical cleavage, thereby blocking expression of the target gene or genes. For example, the gene can be targeted to specific cells to alleviate a genetic abnormality caused by overexpression of a cellular or viral oncogene. In addition, the method can be used to treat negative dominant genetic diseases in which an abnormal gene product interferes with a normal protein. For example, the method can be used to deliver an antisense or ribozyme directed against the abnormal fibrillin produced in Marfan's syndrome or against the abnormal collagen produced in osteogenesis imperfecta type I. The method can also be used to .se expression of the genes of an infecting pathogen such as a virus (hepatitis, HIV) or a parasite such as malaria, trypanosome, lysteria, or mycoplasma. For example, hepatitis genes such as the genes encoding one or more of the surface or core IP~gPCI rae% "9es~SI L WO 94/23050 PCT/US94/03643 -6antigens can be assembled in an expression vector in reverse orientation to generate an antisense transcript which blocks translation of the corresponding genes.

The molecular complex of this invention is adaptable for delivery of a wide range of genes to a specific cell or tissue. In a preferred embodiment, the complex is targeted to the liver by exploiting the hepatic asialoglycoprotein receptor system which allows for in vivo transfection of hepatocytes by the process of receptor-mediated endocytosis.

The method of the invention can be used to treat virus infections of liver cells. This includes infections by any of the liver-spe_: hepatitis viruses. Infection by human hepatitis B virus often results in a chronic, persistent infection. This form of viral infection is more suitably treated by antisense gene therapy compared to antisense oligonucleotide therapy.

Because the hepatitis B virus genome is very small, coding for only three extensively overlapping RNA transcripts, antisense genes can be engineered to encode an RNA that can hybridize to one or more large regions common to all of the viral transcripts. In a preferred embodiment, a complex can be used to deliver a plasmid-borne antisense gene specifically to chronically infected hepatocytes to block the production of hepatitis B virus. The gene can code for the production of an antisense RNA transcript that hybridizes to all of the RNAs produced by the hepatitis B virus, inhibiting production of all viral polypeptides. The resulting soluble complex is administered parenterally to target liver cells of the individual afflicted with the virus in amounts sufficient to selectively transfect the cells and to provide sufficient production of the antisense RNA to achieve inhibition of virus production.

The invention is illustrated further by the following exemplification.

EXEMPLIFATION

Materials and Methods Plasmid DNA Construction The 9.4 Kb plasmid pAdw-HTD, which contains two head-to-tail copies of the hepatitis B virus (HBV) genome, was provided by T.J. Liang (Massachusetts General Hospital, Boston, MA). The first construct termed pJ3l1.0HTD1 or "Anti S" was created by first digesting pAdw-HTD with the restriction endonucleases EcoRI and EcoRV followed by subsequent isolation of 1044bp fragment by agarose gel electrophoresis and glass bead extraction (Geneclean II@, Biol01, LaJolla, CA). This fragment spans most of the pre-S2 signal peptide gene and the complete surface antigen gene but not the surface antigen promoter region. This EcoRI/EcoRV fragment was then ligated into the EcoRI and Smal sites within the polylinker region of the expression vector PJ30 (ATCC, 37719, Nu Acgd Res. 18:1068, 1990) in such a fasion that transcription, driven by the SV40 early promoter, would produce a 1Kb antisense strand of messenger RNA (mRNA) that could then bind to its

IP

s I WO 94/23050 PCT[US94/03643 -7complimentary sequence in the HBV pregenomic mRNA and inihibit translation of HBV surface antigen, thereby disrupting viral assembly (see Figure 1).

The second construct, termed pJ3Q0.8HTD3 or "Anti C" was designed to generate a 0.8 Kb antisense mRNA complementary to the region in the HBV pregenomic mRNA which encodes the precore and core antigens. Core antigen, like the surface antigen, is a coat protein that is essential for viral packaging. Also included in he Anti C fragment is the 1 Ibp direct repeat sequence, denoted DR1, this is critically involved in the initiation of viral DNA synthesis, as well as the unique cleavage/polyaden,;!.ation signal specifying the common 3' termini of all HBV RNA species (Ganem and Varmus (1987) Ann. Rev. Biochem. 56:651- 693). As a result, antisense mRNA transcribed from this region should block all viral protein synthesis as well as replication. To construct pJ320.8HTD3, pAdw-HTD was cleaved with the restriction endonucleases Fspl and Apal to generate a 802bp fragment which was separated by agarose gel electrophoresis and isolated using Geneclean II®. The Anti C fragment was ligated into the Apal/Smal sites within the polylinker region of the cloning vector pGEM-7zf(+) (Promega, Madison, WI). The Anti C fragment was then subcloned into the Clal and Smal sites within the polylinker region of the pJ3f2 vector. To accomplish this, the pGEM clone was first cut with Apal and blunted by the addition of the large Klenow fragment of DNA polymerase I plus 200mM dNTP mixture. Blunting of the Apal end was followed by digestion with Clal to yield a 818bp fragment which was then ligated into the pJ30 expression vector (Figure 1).

Competent DH5ca coli (Gibco BRL) were transformed with the plasmid constructs according to standard protocols (Sambrook et al (1989) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, 2nd Large scale preparations of the plasmid DNA were carried out by standard procedures.

Cells and Cell Culture Human hepatoma, HepG2 .2.15 cells (provided by Dr. George Acs, Albert Einstein College of Medicine, Bronx, NY) were maintained and grown in minimum essential medium (MEM) supplemented with 0.1mM non-essential amino acids, 0.1mM sodiumpyruvate, 2mM L-glutamine, 50 units/ml penicillin, 50ig/ml streptomycin and 10% fetal calf serum. HepG2 .2.15 are clonal cells derived from HepG2 cells transformed with a plasmid containing the HBV genome. The constitutively produce and secrete hepatitis B surface antigen particles, nucleocapsids and virions (Acs t aL (1987) Proc. Natl. Acad. Sci. USA 84:4641-4644).

Preparation ofTargetable Antisense mRNA-Generating Plasmid and Oligonucleotide DNA Antisense plasmid pJ3Q0.8HTD3 and a synthetic 21-mer antisense oligodeoxynucleotide (Synthetic Genetics, San Diego, CA) complimentary to the region in the human hepatitis B virus (ayw subtype) genome that encodes the polyadenylation signal, were titrated with asialoorosomucoid (ASOR)-polylysine conjugates to form soluble $~P(LI~Dll~os~sa~ lr~- WO 94/23050 PCT/US94/03643 -8complexes using an agarose gel retardation assay as described by Wu and Wu (1987) J. Biol. Chem. 262:4429-4432; U.S. Patent No. 5,166,320. For these experiments, a ratio of 0.8:1 by weight (ASOR-polylysine conjugate:DNA), which allowed full retardation of the DNA, was selected and used to prepare the antisense plasmid DNA complex while a ration of 1.6:1 by weight was selected for preparation of the antisense oligonucleotide complex.

Assay of Anitisense Plasmid and Oligonucleotide Activity Delivered to Cells via Receptor- Mediated Endocvtosis To determine the effect on viral gene expression of the antisense mRNA transcribed by the Anti C construct, HepG2 .2.15 cells were seeded at 3.5x10 6 /60mm dish 24 hours prior to use. Preceding transfection, all cells were treated with 100M chloroquine for one hour, followed by 3 washes with phosphate-buffered saline. Cells were then overlaid with fresh MEM containing 100pg of the antisense plasmid in complex or 690lig of antisense oligonucleotide in complex plus enough calcium chloride to increase the Ca concentration 2mM followed by incubation at 37 0 C and 5% CO 2 (a slight modification of the protocol of Wu and Wu (1988) Biochemistry 22:887-892). As controls, some cells were left untransfected, transfected with 100 tg of antisense plasmid DNA alone, or with 690pg of a!ntisense oligonucleotide alone. To determine baseline hepatitis B virus surface antigen (HBsAg) levels, an ELISA (Abbott) assay was performed on 501il samples of medium removed from each plate prior to transfection and assayed according to the procedure described by the manufacturer. 50il samples of medium were subsequently removed from each dish every day for 6 days and processed for HBsAg.

RESULTS

pJ320.8HTD3 was introduced into HepG 2.2.15 cells via asialoglycoprotein receptor mediated endocytosis in order to examine the plasmid's ability to inhibit production of HBsAg. As a reference for comparison some cells were also treated with an antisense 21-mer oligodeoxynucleotide directed against the unique HBV polyadenylation signal sequence.

This oligodeoxynucleotide was idential to the one used by Wu and Wu (1992) J. Biol. Chem.

262:12436-12439 to inhibit HepG 2.2.15 HBsAg expression. A significant reduction in HBsAg expression, relative to untreated cells, was observed in cells that received either pJ300.8HTD3-polylysine-ASOR complex or the antisense oligo-polylysine-ASOR complex (Figure In both cases the inhibition persisted for a least 6 days. Reduction of HBsAg was not seen in cells treated with plasmid or oligodeoxynucleotide alone, suggesting that the ASOR-polylysine conjugate was necessary to deliver these DNAs to their intracellular sites of action. In a separate experiment, cells treated with a pJ321.0HTD 1-complex exhibited a similar reduction in HBsAg levels compared to cells treated with pJ3Q0.8HTD3-complex (data not shown).

-ss~naeaa Iplrrrma~imPe~u~aa~ns~sa~rrr-- WO 94/23050 PCTIUlS94/03643 -9- As figure 2 shows pJ320.8HTD3 was as effective as the antisense oligonucleotide at inhibiting surface antigen expression, both in terms of the level of inhibition as well as its duration. However, the total amount of plasmid added to the cells was almost 7-fold less than that of the anti-HBV oligonucleotide. Calculated on a molar basis, each molecule of pJ320.8HTD3 was roughly equivalent to 1400 molecules of the antisense oligonucleotide in inhibiting HBsAg expression. We expect that this was due to the ability of ite plasmid to generate many copies of its antisense transcript once it was delivered into the cell. It is also possible that differences in stability, site of action, incracellular retention, or differences in the properties of the complexes made with each type of DNA may have contributed to this effect.

This illustrates a potential advantage ofplasmid based antisense systems compared to oligonucleotides. A smaller amount of DNA needs to be delivered to the target eel in order to achieve a therapeutic dose. In addition, a plasmid contains cis-acting sequences, such as promoters, enhances, polyadenylation sites, origins of replication, etc. which directed or influence the expression of its RNA. These sequences can be modified or substituted in order to tailer expression for specific circumstances. For example, one could incorporate an inducible promoter into the plasmid in order to activate expression of the antisense mRNA only at certain times or under certain conditions. Or, one could achieve a very short term, transient inhibition by use of consensus RNA destabilizing elements within the 3' untranslated region of the mRNA. Another possibility would be to produce a sustained antisense mediated inhibition by incorporated sequences into the plasmid which would allow it to be maintained episomally within the cell. Many other characteristics could also be incorporated into a plasmid-based antisense system, thus allowing for a great deal of flexibiltiy and control in its use.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, 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.

~IBA

Claims (33)

1. A soluble molecular complex for targeting a gene encoding an antisense RNA to a specific cell, the complex comprising an expressible gene encoding an RNA which hybridizes to and inhibits the function of a cellular RNA, and a carrier comprising a cell specific binding agent and a gene-binding agent.

2. A soluble complex of claim 1, wherein the antisense RNA is directed against the RNA transcript of a viral or cellular oncogene.

3. A soluble complex of claim 1, wherein the antisense RNA is directed against an RNA transcript of a pathogen.

4. A soluble complex of claim 3, wherein the pathogen is a virus.

A soluble complex of claim 4, wherein the virus is hepatitis.

6. A soluble complex of claim 4, wherein the virus is HIV.

7. P. soluble complex of claim 1, wherein the antisense RNA is directed against a cellular RNA associated with a dominant negative genetic disorder.

8. A soluble complex of claim 1, wherein the antisense RNA is a ribozyme.

9. A soluble molecular complex of claim i, wherein the gene-binding agent is a polycation.

A soluble molecular complex of claim 8, wherein the polycation is polylysine.

11. A soluble molecular complex of claim 1, wherein the cell-specific binding agent binds a surface receptor of the cell which mediates endocytosis.

12. A therapeutic composition comprising a solution of the molecular complex of claim 1 in a physiologically acceptable vehicle.

13. A soluble molecular complex for targeting a gene encoding an antisense RNA to a heDatocyte. the comolex comorising an exoressible gene encoding an antisense RNA AMENDED SHEET I I 1 9 which hybridizes to an RNA transcript produced in the hepatocyte, and a carrier comprising a ligand for the asialoglycoprotein receptor and a polycation.

14. A soluble molecular complex of claim 13, wherein the antisense RNA is directed against an RNA transcript of hepatitis virus.

A soluble molecular complex of claim 13, wherein the polycation is polylysine.

16. A soluble moleculsE complex of claim 13, wherein the gene is contained in an expression vector along with genetic regulatory elements necessary for expression of the gene by the hepatocyte.

17. A soluble molecular complex of claim 16, wherein the expression vector is a plasmid or viral DNA.

18. A soluble molecular complex of claim 13, wherein the antisense RNA is a ribozyme.

19. A therapeutic composition comprising a solution of the molecular complex of claim 13, in a physiologically acceptable vehicle.

A method of blocking translation of an RNA transcript in a cell of an organism, comprising administering to an organism a soluble molecular complex comprising an expressible gene encoding an antisense RNA which hybridizes to and inhibits the function of a cellular RNA, and a carrier comprising a cell-specific binding agent and a gene binding agent.

21. A method of claim 20, wherein the antisense RNA is directed against the RNA transcript of a viral or cellular oncogene.

22. A method of claim 20, wherein the antisense RNA is directed against a transcript of a pathogen.

23. A method of claim 22, wherein the pathogen is hepatitis'virus.

24. A method of claim 20, wherein the gene-binding agent is a polycation. A method of claim 24, wherein the polycation is polylysine.

AjivENDEcD -SiIEET I-I -12-

26. A method of claim 20, wherein the cell-specific binding agent binds a surface receptor of the cell which mediates endocytosis.

27. A method of claim 20, wherein the ligand is an asialoglycopotein and the targeted cell is a hepatocyte.

28. A method of claim 20, wherein the antisense RNA is a ribozyme.

29. A method of claim 20, wherein the molecular complex is administered intravenously.

The soluble complex of claim 5, wherein the hepatitis virus is hepatitis B virus.

31. The soluble complex of claim 14, wherein the hepatitis virus is hepatitis B virus.

32. A soluble molecular complex for targeting a gene encoding an antisense RNA to a specific cell, substantially as herein described with reference to the Exemplification or Figure 1.

33. A method of blocking translation of an RNA transcript in a cell, substantially as herein described with reference to the Exemplification. 15 DATED this 17th day of October, 1997 o UNIVERSITY OF CONNECTICUT AND TARGETECH, INC Attorney: IAN T. ERNST Fellow Institute of Patent Attorneys of Australia of SHELSTON WATERS o *LJJ o' *g ~a

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