CA2159916A1 - Targeted delivery of genes encoding antisense polyribonucleotides - Google Patents
Targeted delivery of genes encoding antisense polyribonucleotidesInfo
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- CA2159916A1 CA2159916A1 CA002159916A CA2159916A CA2159916A1 CA 2159916 A1 CA2159916 A1 CA 2159916A1 CA 002159916 A CA002159916 A CA 002159916A CA 2159916 A CA2159916 A CA 2159916A CA 2159916 A1 CA2159916 A1 CA 2159916A1
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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Abstract
Molecular complexes for targeting a gene encoding an antisense polyribonucleotide to a specific cell in vivo obtaining production of the polyribonucleotide within the targeted cell, and effecting specific inhibition of the expression of cellular or noncellular genes are disclosed. An expressible gene encoding a desired antisense polyribonucleotide is complexed to a conjugate of a cell-specific binding agent and a gene-binding agent. The cell-specific binding agent is specific for a cellular surface structure which mediates internalization of ligands by endocytosis. An example is the asialoglycoprotein receptor of hepatocytes. The gene-binding agent 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 a cell. The molecular complex is stable and soluble in physiological fluids and can be used in antisense gene therapy to selectively transfect cells in vivo or in vitro to provide for production of the antisense polyribonucleotide within the targeted cell, and inhibition of the expression of cellular or noncellular genes.
Description
Wo 94l23050 PCT/US94/03643 TARGFTFT~ nFT TVFRY OF GFNF~ F~COnING
ANTISFNSF POT YF~TRONUCT FOTrnF
5 Rack~rollnll of the Invention ~ nti.e~n~e polynucleotides are a means of specifically inhibiting unwanted gene ~xp~cssion 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 10 RNAs with catalytic activity (ribozymes), which can selectively bind to complementary sequences on a target RNA and physically destroy the target by mPAi~ting a cleavage reaction.
~ ntieen~e polynucleotides can be in the form of small, chemically synth~si7Pcl DNA
or RNA oligonucleo- tides, or can be larger RNAs, such as rnRNAs, biosynthetir~lly 15 generatediny~or~vivoby~.d.l~.;plionofan~nti~n~egene. Sincehundredsofcopies of RNA can be synthPsi7PA from each copy of a gene, a few molecules of an ~nti~çnee 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 ~nti~n~e mRNA to bind with greater affinity and specificity compared to an oligonucleo-20 tide. In addition, unlike oligonucleotides, a plasmid-borne gene can incorporate a wide variety of supplement~l DNA sequences to enh~nce or modulate the expression of the ~ntieenee polyribonucleotide. For example, inclusion of origin sequences which direct episomal replication of a plasmid and promoter and/or enh~ne~r sequences that sustain a high level of ~xl~lession over long periods would allow long term production of an ~nti~erl~e 25 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 ~ntieçn~e oligonucleotide would most likely require continuous or repeated intravenous ~lminietration or highly stable ~nti~n~e constructs.
30 Sl-mm~y of the Tnvention This invention pertains to a soluble molecular complex for L~ g 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 compri~es an t;~lG~sible gene encoding a desired polyribonucleotide complexed to a carrier which is a conjugate of a cell-specific 35 binding agent and a gene-binding agent. In ~nti~ence 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 messen~er 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 2~9916 can be a gene or genes of cellular origin (e.g., a cellular oncogene) or of noncellular origin (e.g., a viral oncogene or the genes of an infecting pathogen such as a virus or a parasite such as m~1~ri~ trypanosome, Iysteria, or mycoplasma).
The cell-specific binding agent is specific for a cellular surface structure, typically a receptor, which mediates intern~li7~tion 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 int~?rn~li7~tion 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 the carrier is stable and soluble in physiological fluids. It can be ~11minietered in vivo where it is selectively taken up by the target cell via the surface-structure-mp~ tec~ endocytotic paLhw~y. The incorporated gene expressed and the gene-encoded product acclln ~ tes within the transfected cell.The soluble molecular complex of this invention can be used to specifically transfect cells in vivo or ~ 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 ~ cssion of cellular or foreign genes. The RNA transcript produced from the delivered gene hybridizes with its complement~ry RNA, inhibiting its function either by steric hindrance, or by physical cleavage, thereby blocking e~ es~ion of the target gene or genes.
Brief Description of the nrawir~e Figure 1 is a schem~tic depiction of the construction of a plasmid encoding an ~ntie~n~e RNA directed against hepatitis B surface antigen (pJ3Q0.8HTDl) and a plasmid encoding an antisense RNA directed against the hepatitis core antigen gene as well as the DRl and polyadenylation site.
Figure 2 shows the reduction of HBsAg mediated by 21-mer ~ntie~nee oligodeoxynucleotide and ~ntieçnee mRNA-generating plasmid pJ3Q0.8HTDl (Anti-C).
net~iled nescription of the Invention 7 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 complex compri.ePs the gene to be delivered complexed to a carrier made up of a binding 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 polyribonucleotide is produced therein.
21S991~
ANTISFNSF POT YF~TRONUCT FOTrnF
5 Rack~rollnll of the Invention ~ nti.e~n~e polynucleotides are a means of specifically inhibiting unwanted gene ~xp~cssion 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 10 RNAs with catalytic activity (ribozymes), which can selectively bind to complementary sequences on a target RNA and physically destroy the target by mPAi~ting a cleavage reaction.
~ ntieen~e polynucleotides can be in the form of small, chemically synth~si7Pcl DNA
or RNA oligonucleo- tides, or can be larger RNAs, such as rnRNAs, biosynthetir~lly 15 generatediny~or~vivoby~.d.l~.;plionofan~nti~n~egene. Sincehundredsofcopies of RNA can be synthPsi7PA from each copy of a gene, a few molecules of an ~nti~çnee 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 ~nti~n~e mRNA to bind with greater affinity and specificity compared to an oligonucleo-20 tide. In addition, unlike oligonucleotides, a plasmid-borne gene can incorporate a wide variety of supplement~l DNA sequences to enh~nce or modulate the expression of the ~ntieenee polyribonucleotide. For example, inclusion of origin sequences which direct episomal replication of a plasmid and promoter and/or enh~ne~r sequences that sustain a high level of ~xl~lession over long periods would allow long term production of an ~nti~erl~e 25 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 ~ntieçn~e oligonucleotide would most likely require continuous or repeated intravenous ~lminietration or highly stable ~nti~n~e constructs.
30 Sl-mm~y of the Tnvention This invention pertains to a soluble molecular complex for L~ g 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 compri~es an t;~lG~sible gene encoding a desired polyribonucleotide complexed to a carrier which is a conjugate of a cell-specific 35 binding agent and a gene-binding agent. In ~nti~ence 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 messen~er 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 2~9916 can be a gene or genes of cellular origin (e.g., a cellular oncogene) or of noncellular origin (e.g., a viral oncogene or the genes of an infecting pathogen such as a virus or a parasite such as m~1~ri~ trypanosome, Iysteria, or mycoplasma).
The cell-specific binding agent is specific for a cellular surface structure, typically a receptor, which mediates intern~li7~tion 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 int~?rn~li7~tion 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 the carrier is stable and soluble in physiological fluids. It can be ~11minietered in vivo where it is selectively taken up by the target cell via the surface-structure-mp~ tec~ endocytotic paLhw~y. The incorporated gene expressed and the gene-encoded product acclln ~ tes within the transfected cell.The soluble molecular complex of this invention can be used to specifically transfect cells in vivo or ~ 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 ~ cssion of cellular or foreign genes. The RNA transcript produced from the delivered gene hybridizes with its complement~ry RNA, inhibiting its function either by steric hindrance, or by physical cleavage, thereby blocking e~ es~ion of the target gene or genes.
Brief Description of the nrawir~e Figure 1 is a schem~tic depiction of the construction of a plasmid encoding an ~ntie~n~e RNA directed against hepatitis B surface antigen (pJ3Q0.8HTDl) and a plasmid encoding an antisense RNA directed against the hepatitis core antigen gene as well as the DRl and polyadenylation site.
Figure 2 shows the reduction of HBsAg mediated by 21-mer ~ntie~nee oligodeoxynucleotide and ~ntieçnee mRNA-generating plasmid pJ3Q0.8HTDl (Anti-C).
net~iled nescription of the Invention 7 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 complex compri.ePs the gene to be delivered complexed to a carrier made up of a binding 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 polyribonucleotide is produced therein.
21S991~
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 a~plo~,iate genetic regulatory elements required for transcription of thc gene by a cellular RNA polymerase and proce~ing of the primary RNA transcript by cellular proteins into a stable form of RNA, such as mRNA. These include promoter and enh~n~er elements c operable in the target cell, as well as other elements such as polyadenylation signals and splicing si~n~l~, which ~letçrmine the intf rn~l 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 10 along with the genetic regulatory elements nece~ y 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 intern~ii7~tion by, for example, the process of endocytosis. The surface structure can be a protein, polypeptide, carbohydrate, lipid or combination thereof. It 15 is typically a surface receptor which me~i~t~s endocytosis of a ligand. Thus, the binding agent can be a natural or synthetic ligand which binds the l~c~lor. The ligand can be a protein, polypeptide, glycoprotein or glycopeptide which has functional groups that are exposed sufficiently to be recogniæd by the cell surface structure. It can also be a component of a biological organism such as a virus, cells (e.g., r~mm~ n, b~rtçri~l, 20 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.
T ig~n(l~ useful in forming the carrier will vary according to the particular cell to be targeted. For ~ge~ g hepatocytes, glycoproteins having exposed If ..llill~i carbohydrate 25 groups such as asialoglyco~lo~eill (galactose-tçrmin~l) 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 e,~ym~Lic desialylation of glycoploteills that possess terminal sialic acid and penllltim~te galactose residues. Alternatively, asialoglycoprotein 30 ligands can be formed by coupling galactose tf rmin~l carbohydrates such as lactose or arabinog~l~ct~n to non-galactose bearing proteins by reductive lactos~",i"~1lion.
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 B12 or bile acids for 35 enterocytes and insulin for fat cells. ~ltern~tively, the cell-specific binding agent can be a receptor or receptor-like molecule, such as an antibody which binds a ligand (e.g., 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 thegene must be sufficiently stable in vivo to prevent signifir~nt uncoupling of the gene 215991~
WO 94l230s0 ; PCT/US94/03643 extracellularly prior to int~m~li7~tion by the target cell. However, the complex is cleavable under ~plo~liate 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 m~teri~le can bind noncovalently with the gene to form a soluble, targetable molecular complex which is stable extracellularly but releasable intracellularly. Suitable polycations are polylysine, poly~gil~ e, polyornithine, basic proteins such as histones, avidin, prota~ es and the like. A ~r~ c;d polycation is polylysine (e.g., ranging from 3,800 to 60,000 daltons). Other noncovalent bonds that can be used to releasably link the ~ e~ible gene include hydrogen bonding, hydrophobic bonding, electrostatic bonding alone or in combination such as, anti-polynucleotide antibodies bound to polynucleotide, and strepavidin or avidin binding to polynucleotide co~ 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 l,r~r~ d linkage is a peptide bond. This can be formed with a water soluble carborliimidç as described by Jung, G. ~ ~L
(1981)Riocht?m Rioehys.Res.Comml~n 101::599-606. An~ltern~tivelinkageisadisulfide bond.
The linkage reaction can be o~lill,i~ed for the particular cell-specific binding agent and gene-binding agent used to form the carrier. Reaction conditions can be ~lesigned to m~imi7~ linkage formation but to ", i "; ", ;,~ the formation of aggregates of the carrier components. The optimal ratio of cell-specific binding agent to gene-binding agent can be detçrmin~d 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 e~ch~nge chromatography (e.g., AquaporeTM cation ~xrh~nge, Rainin).
In one embodiment, asialoorosomucoid-polylysine conjugate is formed with the croeelinkinp agent 1-(3-dimethylaminopropyl)-3-ethyl carbor~iimi~le After dialysis, the conjugate is separated from unconjugated components by plGp~dli~e acid-urea polyacrylamide gel electrophoresis (pH 4-5). The conjugate can be further purified on the carboxymethyl functionalized column (Waters AP-l column). See U.S. Patent Application Serial No. 08/043,008, filed on April 5, 1992, the te~chinge of which are incorporated by reference herein.
The gene encoding the ~ntie~nee construct can be complexed to the carrier by a stepwise dialysis procedure. In a pre~ll~d method, for use with carriers made of polycations 21599~5 such as polylysine, the dialysis procedure begins with a 2M NaCl dialyzate and ends with a .15M NaCl solution. The gradually decreasing NaCl 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 neces~y; the gene and carrier are simply mixed and S incubated.
The molecular complex can contain more than one copy of the same gene or one or more different genes. Preferably, the weight ratio of gene to the carrier is from about 1 :5 to S: 1, preferably about 1 :2 (approximate molar ratio 1: 100 to 1 :200). The a~propllate ratio for a particular polynucleotide and carrier can be cl~ ed by the gel retardation assay described in U.S. Patent No. 5,166,320.
The molecular complex of this invention can be ~(lminietçred ~ellleldlly.
Preferably, it is injected intravenously. The complex is ~-lmini~tçred in solution in a physiologically acceptable vehicle.
Cells can be transfected in ~i~Q for transient production of the polyribonucleotide.
15 For prolonged production, the gene can be ~rlmini.~t~red repeatedly. ~ltern~tively, the transfected target cell can be stim~ tecl to replicate by surgical or ph~rrn~(~ological 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 tç~chin~;s of which are incorporated by reference herein. Drugs that disrupt translocation or fusion of endosomes to Iysosomes such 20 as colchicine or taxol can be used to prolong ex~;,sion. See U.S. Patent Application Serial No. 950,789, filed September 24, 1992, the tç~chin~ of which are incorporated by ~c;felGllce 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 25 te~chin~ of which are incorporated by reference herein.
The method of this invention can be used to selectively deliver a gene to a target cell in YiVo for ~nti~çn~e gene therapy or other applications which require inhibition of the ~x~ ssion of specific cellular or foreign genes. The RNA transcript produced from the delivered gene hybridizes with its complement~ry RNA, inhibiting its function either by 30 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 ovel~x~lession of a cellular or viral oncogene. In addition, the method can be used to treat negative dominant genetic ~ e~ees in which an abnormal gene product in~ r~lt;s with a normal protein. For example, the method can be used to deliver an 35 ~nti~çn~e or ribozyme directed against ~e 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 inhibit the expression of the genes of an infecting pathogen such as a virus (hepatitis, HIV) or a parasite such as m~l~ri~, trypanosome, Iysteria, or mycoplasma. For example, hepatitis genes such as the genes encoding one or more of the surface or core -antigens can be assembled in an ~ ssion vector in reverse orientation to generate an antisense kanscript which blocks tr~ncl~tion 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 ~l~r~ d embodiment, the complex is targeted to the 5 liver by exploiting the hepatic asialoglycoprotein receptor system which allows for in ViVO
kansfection of hepatocytes by the process of receptor-me~ te~l endocytosis.
The method of the invention can be used to keat virus infections of liver cells. This includes infections by any of the liver-specific hepatitis viruses. Infection by human hep~titic B virus often results in a chronic, persictent infection. This form of viral infection is more 10 suitably keated by ~nti~n~e gene therapy compared to ~nti~nce oligonucleotide therapy.
Because the hepatitis B virus genome is very small, coding for only three extensively overlapping RNA transcripts, ~nti~nce genes can be e~gin~ered to encode an RNA that can hybridize to one or more large regions common to all of the viral kanscripts. In a pler~ d embodiment, a complex can be used to deliver a plasmid-borne ~nticen~e gene specifically to 15 chronically infected hepatocytes to block the production of hepatitis B virus. The gene can code for the production of an ~nti~n~e RNA transcript that hybridizes to all of the RNAs produced by the hepatitis B virus, inhibiting production of all viral polypeptides. The resllltin~ soluble complex is ~rlmini~tered parenterally to target liver cells of the individual afflicted with the virus in amounts sufficient to selectively transfect the cells and to provide 20 sufficient production of the ~nti~Pn~e RNA to achieve inhibition of virus production.
The invention is illustrated further by the following exemplification.
~X~MPI ,T~CATION
Materials ~nd Methods Pl~mid nNA Co~truction The 9.4 Kb plasmid pAdw-HTD, which contains two head-to-tail copies of the 30 hepatitis B virus (HBV) genome, was provided by T.J. Liang (l~ .C~I~`i General Hospital, Boston, MA). The first construct termed pJ3Ql.OHTDl or "Anti S" was created by first digesting pAdw-HTD with the restriction endonucleases EcoRI and EcoRV followed by subsequent isolation of 1 044bp fragment by agarose gel electrophoresis and glass bead extraction (Geneclean II~), Biol 01, LaJolla, CA). This fragment spans most of the pre-S2 35 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 ofthe t;~ s~ion vector PJ3Q (ATCC, 37719, Nuc. Acids Res. 1~:1068, 1990) in such a fasion that transcription, driven by the SV40 early promoter, would produce a lKb ~nti~çn~e strand of mess~n~er RNA (mRNA) that could then bind to its 2159?1~
WO 9412~,050 PCT/US94/03643 complimentary 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 ~nti~Pn~e rnRNA 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 p~r.k~ping Also included in he Anti C fragment is the 1 lbp direct repeat sequence, denoted DR1, this is critically involved in the initiation of viral DNA
synthesi.~, as well as the unique cleavage/polyadenylation signal specifying the common 3' termini of all HBV RNA species (Ganem and Varmus (1987) Ann Rev. Biochem ~:651-693). As a result, anti~en~e mRNA transcribed from this region should block all viral protein synthesis as well as replication. To construct pJ3Q0.8HTD3, 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~g). 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 pJ3Q 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 pJ3Q expression vector (Figure 1).
Competent DHSa P çoli (Gibco BRL) were transformed with the plasmid constructs according to standard protocols (Sambrook çt ~L (1989) Molec~ r Cloni~ - ~
r ~horatory M~nual~ Cold Spring Harbor Laboratory, 2nd ed.). Large scale p~c~Lions of the plasmid DNA were carried out by standard procedures.
Cells ~n-l Cell Culture Hurnan hep~tom~, HepG2 .2.15 cells (provided by Dr. George Acs, Albert Einstein College of Medicine, Bronx, NY) were m~int~in~c~ and grown in ~ llll es~çnti~l medium (MEM) supplementecl with 0.1mM non-essential arnino acids, 0.1mM sodiunl~y,,l~ate, 2mM
L-glutamine, 50 units/ml penicillin, 50~Lg/ml ,ll~tomycin and 10% fetal calf serum. HepG2 .2.15 are clonal cells derived from HepG2 cells transforme-d with a plasmid co~ the HBV genome. The constitutively produce and secrete hepatitis B surface antigen particles, nucleocapsids and virions (Acs et ~L (1987) Proc. Natl. Acad. Sci. USA 84:4641-4644).
P~ ion of T~r~etable Anti~en~o mRNA-Gener~tir~ Pl~mid ~n~l Ol~on~ leotide DNA
~nti.~n~e plasmid pJ3Q0.8HTD3 and a synthetic 21-mer ~nti~en~e oligodeoxynucleotide (Synthetic Genetics, San Diego, CA) compliment~ry 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 21~9916 WO 94123050 ~ PCT/US94/03643 complexes using an agarose gel retardation assay as described by Wu and Wu (1987) J. Riol. Chenl ~:4429-4432; U.S. Patent No. 5,166,320. For these ~ ent.~, 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 p.~aldlion of the ~nti~en~e oligonucleotide complex.
A~ v of Aniti~n~e Pl~mid ~nl1 Oli~onl~rleotide Activitv Delivered to Cell~ via Receptor-~e~i~t~-1 Fntl-~cytosi~
To determine the effect on viral gene ~Lession of the ~nti~en~e mRNA transcribedby the Anti C construct, HepG2 .2.15 cells were seeded at 3.5xl06/60mm dish 24 hours prior to use. Prece(ling transfection, all cells were treated with 100,uM chloroquine for one hour, followed by 3 washes with phosphate-buffered saline. Cells were then overlaid with fresh MEM co~ 100,ug of the ~nti~Pn~e plasmid in complex or 690,ug of ~nti~en~e oligonucleotide in complex plus enough calcium chloride to increase the Ca~ concentration 2mM followed by incubation at 37C and 5% CO2 (a slight modification of the protocol of Wu and Wu (1988) Rio~hemi~trv ~Z:887-892). As controls, some cells were left untransfected, transfected with 100~1g of ~nti~en~e plasmid DNA alone, or with 690~g of ~nti~?n~e oligonucleotide alone. To determine baseline hepatitis B virus surface antigen (HBsAg) levels, an ELISA (Abbott) assay was performed on 50,u1 samples of mediumremoved from each plate prior to transfection and assayed according to the procedure described by the m~nllf~tllrer. 50,u1 samples of medium were subsequently removed from each dish every day for 6 days and processed for HBsAg.
RF~UT TS
pJ3Q0.8HTD3 was introduced into HepG 2.2.15 cells via asialoglyco~roteil, receptor mediated endocytosis in order to e~min~. 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. Riol. Chem ~1.:12436-12439 to inhibit HepG 2.2.15 HBsAg exp.es~ion. A ~ignifi~nt reduction in HBsAg c;~æsion~ relative to ullLI ;;aled cells, was observed in cells that received either pJ3Q0.8HTD3-polylysine-ASOR complex or the ~nti~en~e oligo-polylysine-ASOR complex (Figure 2). 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 neces~ to deliver these DNAs to their intracellular sites of action. In a separate t;~lhllent, cells treated with a pJ3Ql .OHTDl-complex exhibited a similar reduction in HBsAg levels colllpaLed to cells treated with pJ3Q0.8HTD3-complex (data not shown).
~t5~91~
~WO 94/23050 PCTIUS94/03643 g As figure 2 shows pJ3Q0.8HTD3 was as effective as the antisense oligonucleotide at inhibiting surface antigen ~plcssion, 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 pJ3Q0.8HTD3 was roughly equivalent to 1400 molecules of the antisense oligonucleotide in inhibiting HBsAg expression. We expect that this was due to the ability of the plasmid to generate many copies of its ~ntieçnee transcript once it was delivered into the cell. It is also possible that differences in stability, site of action, intracellular retention, or differences in the properties of the complexes made with each type of DNA may have contributed to this effect.
This illustrates apotential advantage of plasmid based ~ntiee~ee systems colllpaled to oligonucleotides. A smaller amount of DNA needs to be delivered to the target cel in order to achieve a therapeutic dose. In addition, a plasmid contains cis-acting sequences, such as promoters, enh~n~es, 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 circ~lmet~n~ee~ For çx~mI~le, one could incorporate an inducible promoter into the plasmid in order to activate ~ s~ion of the ~nti.ePnee 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 s~st~inPd ~ntieenee medi~ted inhibition by incorporated sequences into the plasmid which would allow it to be m~int~ined 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.
Fquiv~lente Those skilled in the art will recognize, or be able to ascertain using no more than routine experiment~tion, 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.
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 a~plo~,iate genetic regulatory elements required for transcription of thc gene by a cellular RNA polymerase and proce~ing of the primary RNA transcript by cellular proteins into a stable form of RNA, such as mRNA. These include promoter and enh~n~er elements c operable in the target cell, as well as other elements such as polyadenylation signals and splicing si~n~l~, which ~letçrmine the intf rn~l 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 10 along with the genetic regulatory elements nece~ y 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 intern~ii7~tion by, for example, the process of endocytosis. The surface structure can be a protein, polypeptide, carbohydrate, lipid or combination thereof. It 15 is typically a surface receptor which me~i~t~s endocytosis of a ligand. Thus, the binding agent can be a natural or synthetic ligand which binds the l~c~lor. The ligand can be a protein, polypeptide, glycoprotein or glycopeptide which has functional groups that are exposed sufficiently to be recogniæd by the cell surface structure. It can also be a component of a biological organism such as a virus, cells (e.g., r~mm~ n, b~rtçri~l, 20 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.
T ig~n(l~ useful in forming the carrier will vary according to the particular cell to be targeted. For ~ge~ g hepatocytes, glycoproteins having exposed If ..llill~i carbohydrate 25 groups such as asialoglyco~lo~eill (galactose-tçrmin~l) 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 e,~ym~Lic desialylation of glycoploteills that possess terminal sialic acid and penllltim~te galactose residues. Alternatively, asialoglycoprotein 30 ligands can be formed by coupling galactose tf rmin~l carbohydrates such as lactose or arabinog~l~ct~n to non-galactose bearing proteins by reductive lactos~",i"~1lion.
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 B12 or bile acids for 35 enterocytes and insulin for fat cells. ~ltern~tively, the cell-specific binding agent can be a receptor or receptor-like molecule, such as an antibody which binds a ligand (e.g., 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 thegene must be sufficiently stable in vivo to prevent signifir~nt uncoupling of the gene 215991~
WO 94l230s0 ; PCT/US94/03643 extracellularly prior to int~m~li7~tion by the target cell. However, the complex is cleavable under ~plo~liate 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 m~teri~le can bind noncovalently with the gene to form a soluble, targetable molecular complex which is stable extracellularly but releasable intracellularly. Suitable polycations are polylysine, poly~gil~ e, polyornithine, basic proteins such as histones, avidin, prota~ es and the like. A ~r~ c;d polycation is polylysine (e.g., ranging from 3,800 to 60,000 daltons). Other noncovalent bonds that can be used to releasably link the ~ e~ible gene include hydrogen bonding, hydrophobic bonding, electrostatic bonding alone or in combination such as, anti-polynucleotide antibodies bound to polynucleotide, and strepavidin or avidin binding to polynucleotide co~ 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 l,r~r~ d linkage is a peptide bond. This can be formed with a water soluble carborliimidç as described by Jung, G. ~ ~L
(1981)Riocht?m Rioehys.Res.Comml~n 101::599-606. An~ltern~tivelinkageisadisulfide bond.
The linkage reaction can be o~lill,i~ed for the particular cell-specific binding agent and gene-binding agent used to form the carrier. Reaction conditions can be ~lesigned to m~imi7~ linkage formation but to ", i "; ", ;,~ the formation of aggregates of the carrier components. The optimal ratio of cell-specific binding agent to gene-binding agent can be detçrmin~d 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 e~ch~nge chromatography (e.g., AquaporeTM cation ~xrh~nge, Rainin).
In one embodiment, asialoorosomucoid-polylysine conjugate is formed with the croeelinkinp agent 1-(3-dimethylaminopropyl)-3-ethyl carbor~iimi~le After dialysis, the conjugate is separated from unconjugated components by plGp~dli~e acid-urea polyacrylamide gel electrophoresis (pH 4-5). The conjugate can be further purified on the carboxymethyl functionalized column (Waters AP-l column). See U.S. Patent Application Serial No. 08/043,008, filed on April 5, 1992, the te~chinge of which are incorporated by reference herein.
The gene encoding the ~ntie~nee construct can be complexed to the carrier by a stepwise dialysis procedure. In a pre~ll~d method, for use with carriers made of polycations 21599~5 such as polylysine, the dialysis procedure begins with a 2M NaCl dialyzate and ends with a .15M NaCl solution. The gradually decreasing NaCl 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 neces~y; the gene and carrier are simply mixed and S incubated.
The molecular complex can contain more than one copy of the same gene or one or more different genes. Preferably, the weight ratio of gene to the carrier is from about 1 :5 to S: 1, preferably about 1 :2 (approximate molar ratio 1: 100 to 1 :200). The a~propllate ratio for a particular polynucleotide and carrier can be cl~ ed by the gel retardation assay described in U.S. Patent No. 5,166,320.
The molecular complex of this invention can be ~(lminietçred ~ellleldlly.
Preferably, it is injected intravenously. The complex is ~-lmini~tçred in solution in a physiologically acceptable vehicle.
Cells can be transfected in ~i~Q for transient production of the polyribonucleotide.
15 For prolonged production, the gene can be ~rlmini.~t~red repeatedly. ~ltern~tively, the transfected target cell can be stim~ tecl to replicate by surgical or ph~rrn~(~ological 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 tç~chin~;s of which are incorporated by reference herein. Drugs that disrupt translocation or fusion of endosomes to Iysosomes such 20 as colchicine or taxol can be used to prolong ex~;,sion. See U.S. Patent Application Serial No. 950,789, filed September 24, 1992, the tç~chin~ of which are incorporated by ~c;felGllce 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 25 te~chin~ of which are incorporated by reference herein.
The method of this invention can be used to selectively deliver a gene to a target cell in YiVo for ~nti~çn~e gene therapy or other applications which require inhibition of the ~x~ ssion of specific cellular or foreign genes. The RNA transcript produced from the delivered gene hybridizes with its complement~ry RNA, inhibiting its function either by 30 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 ovel~x~lession of a cellular or viral oncogene. In addition, the method can be used to treat negative dominant genetic ~ e~ees in which an abnormal gene product in~ r~lt;s with a normal protein. For example, the method can be used to deliver an 35 ~nti~çn~e or ribozyme directed against ~e 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 inhibit the expression of the genes of an infecting pathogen such as a virus (hepatitis, HIV) or a parasite such as m~l~ri~, trypanosome, Iysteria, or mycoplasma. For example, hepatitis genes such as the genes encoding one or more of the surface or core -antigens can be assembled in an ~ ssion vector in reverse orientation to generate an antisense kanscript which blocks tr~ncl~tion 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 ~l~r~ d embodiment, the complex is targeted to the 5 liver by exploiting the hepatic asialoglycoprotein receptor system which allows for in ViVO
kansfection of hepatocytes by the process of receptor-me~ te~l endocytosis.
The method of the invention can be used to keat virus infections of liver cells. This includes infections by any of the liver-specific hepatitis viruses. Infection by human hep~titic B virus often results in a chronic, persictent infection. This form of viral infection is more 10 suitably keated by ~nti~n~e gene therapy compared to ~nti~nce oligonucleotide therapy.
Because the hepatitis B virus genome is very small, coding for only three extensively overlapping RNA transcripts, ~nti~nce genes can be e~gin~ered to encode an RNA that can hybridize to one or more large regions common to all of the viral kanscripts. In a pler~ d embodiment, a complex can be used to deliver a plasmid-borne ~nticen~e gene specifically to 15 chronically infected hepatocytes to block the production of hepatitis B virus. The gene can code for the production of an ~nti~n~e RNA transcript that hybridizes to all of the RNAs produced by the hepatitis B virus, inhibiting production of all viral polypeptides. The resllltin~ soluble complex is ~rlmini~tered parenterally to target liver cells of the individual afflicted with the virus in amounts sufficient to selectively transfect the cells and to provide 20 sufficient production of the ~nti~Pn~e RNA to achieve inhibition of virus production.
The invention is illustrated further by the following exemplification.
~X~MPI ,T~CATION
Materials ~nd Methods Pl~mid nNA Co~truction The 9.4 Kb plasmid pAdw-HTD, which contains two head-to-tail copies of the 30 hepatitis B virus (HBV) genome, was provided by T.J. Liang (l~ .C~I~`i General Hospital, Boston, MA). The first construct termed pJ3Ql.OHTDl or "Anti S" was created by first digesting pAdw-HTD with the restriction endonucleases EcoRI and EcoRV followed by subsequent isolation of 1 044bp fragment by agarose gel electrophoresis and glass bead extraction (Geneclean II~), Biol 01, LaJolla, CA). This fragment spans most of the pre-S2 35 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 ofthe t;~ s~ion vector PJ3Q (ATCC, 37719, Nuc. Acids Res. 1~:1068, 1990) in such a fasion that transcription, driven by the SV40 early promoter, would produce a lKb ~nti~çn~e strand of mess~n~er RNA (mRNA) that could then bind to its 2159?1~
WO 9412~,050 PCT/US94/03643 complimentary 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 ~nti~Pn~e rnRNA 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 p~r.k~ping Also included in he Anti C fragment is the 1 lbp direct repeat sequence, denoted DR1, this is critically involved in the initiation of viral DNA
synthesi.~, as well as the unique cleavage/polyadenylation signal specifying the common 3' termini of all HBV RNA species (Ganem and Varmus (1987) Ann Rev. Biochem ~:651-693). As a result, anti~en~e mRNA transcribed from this region should block all viral protein synthesis as well as replication. To construct pJ3Q0.8HTD3, 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~g). 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 pJ3Q 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 pJ3Q expression vector (Figure 1).
Competent DHSa P çoli (Gibco BRL) were transformed with the plasmid constructs according to standard protocols (Sambrook çt ~L (1989) Molec~ r Cloni~ - ~
r ~horatory M~nual~ Cold Spring Harbor Laboratory, 2nd ed.). Large scale p~c~Lions of the plasmid DNA were carried out by standard procedures.
Cells ~n-l Cell Culture Hurnan hep~tom~, HepG2 .2.15 cells (provided by Dr. George Acs, Albert Einstein College of Medicine, Bronx, NY) were m~int~in~c~ and grown in ~ llll es~çnti~l medium (MEM) supplementecl with 0.1mM non-essential arnino acids, 0.1mM sodiunl~y,,l~ate, 2mM
L-glutamine, 50 units/ml penicillin, 50~Lg/ml ,ll~tomycin and 10% fetal calf serum. HepG2 .2.15 are clonal cells derived from HepG2 cells transforme-d with a plasmid co~ the HBV genome. The constitutively produce and secrete hepatitis B surface antigen particles, nucleocapsids and virions (Acs et ~L (1987) Proc. Natl. Acad. Sci. USA 84:4641-4644).
P~ ion of T~r~etable Anti~en~o mRNA-Gener~tir~ Pl~mid ~n~l Ol~on~ leotide DNA
~nti.~n~e plasmid pJ3Q0.8HTD3 and a synthetic 21-mer ~nti~en~e oligodeoxynucleotide (Synthetic Genetics, San Diego, CA) compliment~ry 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 21~9916 WO 94123050 ~ PCT/US94/03643 complexes using an agarose gel retardation assay as described by Wu and Wu (1987) J. Riol. Chenl ~:4429-4432; U.S. Patent No. 5,166,320. For these ~ ent.~, 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 p.~aldlion of the ~nti~en~e oligonucleotide complex.
A~ v of Aniti~n~e Pl~mid ~nl1 Oli~onl~rleotide Activitv Delivered to Cell~ via Receptor-~e~i~t~-1 Fntl-~cytosi~
To determine the effect on viral gene ~Lession of the ~nti~en~e mRNA transcribedby the Anti C construct, HepG2 .2.15 cells were seeded at 3.5xl06/60mm dish 24 hours prior to use. Prece(ling transfection, all cells were treated with 100,uM chloroquine for one hour, followed by 3 washes with phosphate-buffered saline. Cells were then overlaid with fresh MEM co~ 100,ug of the ~nti~Pn~e plasmid in complex or 690,ug of ~nti~en~e oligonucleotide in complex plus enough calcium chloride to increase the Ca~ concentration 2mM followed by incubation at 37C and 5% CO2 (a slight modification of the protocol of Wu and Wu (1988) Rio~hemi~trv ~Z:887-892). As controls, some cells were left untransfected, transfected with 100~1g of ~nti~en~e plasmid DNA alone, or with 690~g of ~nti~?n~e oligonucleotide alone. To determine baseline hepatitis B virus surface antigen (HBsAg) levels, an ELISA (Abbott) assay was performed on 50,u1 samples of mediumremoved from each plate prior to transfection and assayed according to the procedure described by the m~nllf~tllrer. 50,u1 samples of medium were subsequently removed from each dish every day for 6 days and processed for HBsAg.
RF~UT TS
pJ3Q0.8HTD3 was introduced into HepG 2.2.15 cells via asialoglyco~roteil, receptor mediated endocytosis in order to e~min~. 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. Riol. Chem ~1.:12436-12439 to inhibit HepG 2.2.15 HBsAg exp.es~ion. A ~ignifi~nt reduction in HBsAg c;~æsion~ relative to ullLI ;;aled cells, was observed in cells that received either pJ3Q0.8HTD3-polylysine-ASOR complex or the ~nti~en~e oligo-polylysine-ASOR complex (Figure 2). 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 neces~ to deliver these DNAs to their intracellular sites of action. In a separate t;~lhllent, cells treated with a pJ3Ql .OHTDl-complex exhibited a similar reduction in HBsAg levels colllpaLed to cells treated with pJ3Q0.8HTD3-complex (data not shown).
~t5~91~
~WO 94/23050 PCTIUS94/03643 g As figure 2 shows pJ3Q0.8HTD3 was as effective as the antisense oligonucleotide at inhibiting surface antigen ~plcssion, 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 pJ3Q0.8HTD3 was roughly equivalent to 1400 molecules of the antisense oligonucleotide in inhibiting HBsAg expression. We expect that this was due to the ability of the plasmid to generate many copies of its ~ntieçnee transcript once it was delivered into the cell. It is also possible that differences in stability, site of action, intracellular retention, or differences in the properties of the complexes made with each type of DNA may have contributed to this effect.
This illustrates apotential advantage of plasmid based ~ntiee~ee systems colllpaled to oligonucleotides. A smaller amount of DNA needs to be delivered to the target cel in order to achieve a therapeutic dose. In addition, a plasmid contains cis-acting sequences, such as promoters, enh~n~es, 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 circ~lmet~n~ee~ For çx~mI~le, one could incorporate an inducible promoter into the plasmid in order to activate ~ s~ion of the ~nti.ePnee 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 s~st~inPd ~ntieenee medi~ted inhibition by incorporated sequences into the plasmid which would allow it to be m~int~ined 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.
Fquiv~lente Those skilled in the art will recognize, or be able to ascertain using no more than routine experiment~tion, 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 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.
5. A soluble complex of claim 4, wherein the virus is hepatitis.
6. A soluble complex of claim 4, wherein the virus is HIV.
7. A 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 1, wherein the gene-binding agent is a polycation.
10. 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 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.
14. A soluble molecular complex of claim 13, wherein the antisense RNA is directed against an RNA transcript of hepatitis virus.
15. A soluble molecular complex of claim 13, wherein the polycation is polylysine.
16. A soluble molecular complex of claim 13, wherein the gene is contained in anexpression 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.
20. 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.
21. 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.
25. A method of claim 24, wherein the polycation is polylysine.
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 asialoglycoprotein 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.
30. 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.
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.
5. A soluble complex of claim 4, wherein the virus is hepatitis.
6. A soluble complex of claim 4, wherein the virus is HIV.
7. A 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 1, wherein the gene-binding agent is a polycation.
10. 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 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.
14. A soluble molecular complex of claim 13, wherein the antisense RNA is directed against an RNA transcript of hepatitis virus.
15. A soluble molecular complex of claim 13, wherein the polycation is polylysine.
16. A soluble molecular complex of claim 13, wherein the gene is contained in anexpression 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.
20. 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.
21. 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.
25. A method of claim 24, wherein the polycation is polylysine.
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 asialoglycoprotein 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.
30. 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.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US4294393A | 1993-04-05 | 1993-04-05 | |
| US08/042,943 | 1993-04-05 | ||
| PCT/US1994/003643 WO1994023050A1 (en) | 1993-04-05 | 1994-04-04 | Targeted delivery of genes encoding antisense polyribonucleotides |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2159916A1 true CA2159916A1 (en) | 1994-10-13 |
Family
ID=21924584
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002159916A Abandoned CA2159916A1 (en) | 1993-04-05 | 1994-04-04 | Targeted delivery of genes encoding antisense polyribonucleotides |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0693130A1 (en) |
| JP (1) | JPH08508645A (en) |
| AU (1) | AU686614B2 (en) |
| CA (1) | CA2159916A1 (en) |
| WO (1) | WO1994023050A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996021036A2 (en) * | 1994-12-30 | 1996-07-11 | Chiron Viagene, Inc. | Nucleic acid condensing agents with reduced immunogenicity |
| US5780009A (en) * | 1995-01-20 | 1998-07-14 | Nexia Biotechnologies, Inc. | Direct gene transfer into the ruminant mammary gland |
| US6673914B1 (en) | 1998-01-22 | 2004-01-06 | John Wayne Cancer Institute | Human tumor-associated gene |
| AU1480801A (en) * | 1999-11-12 | 2001-06-06 | Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The | Compositions and methods for the diminution or elimination of various cancers |
| WO2004045491A2 (en) | 2002-11-18 | 2004-06-03 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Targeted double stranded rna mediated cell killing |
| EP2069375B1 (en) | 2006-09-28 | 2012-03-28 | Loma Linda University | Apoptotic cell-mediated transfection of mammalian cells with interfering RNA |
| JP6245570B2 (en) * | 2013-10-08 | 2017-12-13 | 国立研究開発法人理化学研究所 | Trypanosoma-related disease therapeutic agent, trypanosome protozoan killing method and use thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992005250A1 (en) * | 1990-09-25 | 1992-04-02 | University Of Connecticut | Prolonging expression of polynucleotides introduced into a cell |
| AU662304B2 (en) * | 1990-10-22 | 1995-08-31 | Fox Chase Cancer Center | DNA construct for providing RNA therapy |
| KR100252547B1 (en) * | 1991-09-05 | 2000-09-01 | 프레드 마리얀스키 | Targeted delivery of poly- or oligonucleotides to cells |
-
1994
- 1994-04-04 JP JP6522433A patent/JPH08508645A/en active Pending
- 1994-04-04 EP EP94912389A patent/EP0693130A1/en not_active Withdrawn
- 1994-04-04 AU AU64978/94A patent/AU686614B2/en not_active Ceased
- 1994-04-04 WO PCT/US1994/003643 patent/WO1994023050A1/en not_active Application Discontinuation
- 1994-04-04 CA CA002159916A patent/CA2159916A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| EP0693130A1 (en) | 1996-01-24 |
| JPH08508645A (en) | 1996-09-17 |
| WO1994023050A1 (en) | 1994-10-13 |
| AU686614B2 (en) | 1998-02-12 |
| AU6497894A (en) | 1994-10-24 |
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