OA17017A - Compositions for targeted delivery of SIRNA. - Google Patents

Abstract

The present invention is directed compositions for targeted delivery of RNA interference (RNAi) polynucleotides to hepatocytes in vivo. Targeted RNAi polynucleotides are administered together with cotargeted delivery polymers. Delivery polymers provide membrane penetration function for movement of the RNAi polynucleotides from outside the cell to inside the cell. Reversible modification provides physiological responsiveness to the delivery polymers.

Description

The delivery of polynucleotide and other substantially cell membrane Imperméable compounds Into a living cell is hlghly restrlcted by the complex membrane System of the cell. Drugs used In antisense, RNAI, and gene thérapies are relatively large hydrophilic polymers and are frequently hlghly negatively charged. Both of these physlcal characteristics preclude their direct diffusion across the cell membrane. For this reason, the major barrier to polynucleotide delivery is the delivery of the polynucleotide across a cell membrane to the cell cytoplasm or nucléus.

One means that has been used to deiiver small nucleic acid in vivo has been to attach the nuclelc acid to either a small targeting molécule or a llpid or sterol. While sonne delivery and activity has been observed with these conjugates, the nucleic acid dose required with these methods has been prohibîtively large.

Numerous transfection reagents hâve been developed that achieve reasonably efficient delivery of polynucleotides to celis in vitro. However, in vivo delivery of polynucleotides using these same transfection reagents is complicated and rendered ineffective by in vivo toxicity, sérum interactions, and poor targeting. Transfection reagents that work well in vitro, cationlc polymers and lipids, typically form large electrostatic particles and destabilize cell membranes. The positive charge of in vitro transfection reagents facilitâtes association with nucleic acid via charge-charge (electrostatic) interactions thus forming the nucleic acid/transfection reagent complex. Positive charge Is also bénéficiai for nonspecific binding of the vehicle to the cell and for membrane fusion, destabilizatlon, or disruption. Destabilization of membranes facilitâtes delivery of the substantially cell membrane imperméable polynucleotide across .a cell membrane. While these properties facilitate nuclelc acid transfer in vitro, they cause toxicity and ineffective targeting in vivo. Cationic charge results in interaction with sérum components, which causes destabilization of the polynucleotide-transfection reagent interaction and poor bioavailability and targeting. Membrane activity of transfection reagents, which can be effective in vitro, often leads to toxicity in vivo.

For in vivo delivery, the vehicle (nucleic acid and associated delivery agent) should be small, less than 100 nm In diameter, and preferably less than 50 nm. Even smaller complexes, less that 20 nm or less than 10 nm would be more useful yet. Delivery vehicles larger than 100 nm hâve very little access to cells other than blood vessel cells in vivo. Complexes formed by electrostatic interactions tend to aggregate or fall apart when exposed to physiological sait concentrations or sérum components. Further, catlonic charge on in vivo delivery vehicles leads to adverse sérum interactions and therefore poor bioavailability. Interestingly, high négative charge can also inhibit in vivo delivery by interfering with interactions necessary for targeting. Thus, near neutral vehicles are desired for In vivo distribution and targeting. Without careful régulation, membrane disruption or destabilizatlon activities are toxic when used In vivo. Baiancing vehicie toxicity with nucleic acid delivery is more easily attained in vitro than in vivo.

Rozema et al., In U.S. Patent Publication 20040162260 demonstrated a means to reversibly regulate membrane disruptive activity of a membrane active polyamlne. The membrane active polyamine provided a means of disrupting cell membranes. pH-dependent réversible régulation provided a means to limit activity to the endosomes of target cells, thus limiting toxicity. Their method relied on modification of amines on a polyamine with 2-propionic-3-methylmaleic anhydride.

This modification converted the poiycation to a poiyanion via conversion of primary amines to pairs of carboxyi groups (β carboxyl and γ carboxyl) and reversibly inhibited membrane activity of the polyamine. Rozema et al. (Bioconjugate Chem. 2003, 14, 51-57) reported that the β carboxyl did not exhibit a full apparent négative charge and by itself was not able to inhibit membrane activity. The addition of the γ carboxyl group was reported to be necessary for effective membrane activity Inhibition. To enable co-delivery of the nucleic acid with the delivery vehicie, the nucleic acid was covalently iinked to the delivery polymer. They were able to show delivery of polynucleotides to cells In vitro using their biologically labile conjugate delivery System. However, because the vehicie was hlghly negatively charged, with both the nucleic acid and the modified polymer havlng high négative charge density, this System was not efficient for in v/vo delivery. The négative charge iikely inhibited cell-specific targeting and enhanced non-specffîc uptake by the réticuloendothélial system (RES). Also using the 2proplonic-3-methylmaleic anhydride-modified polymère, Rozema et al. demonstrated formation of small ternary electrostatic complexes of nucleic acids, polycations, and 2-propionic-3methylmaleic anhydride-modified polymère.

Rozema et al., in U.S. Patent Publication 20080152661, Improved on the method of U.S. Patent Publication 20040162260 by elimlnating the high négative charge density of the modified membrane active polymer. By substituting neutrai hydrophilic targeting (galactose) and steric stabilizing (PEG) groups for the y carboxyl of 2-proptonic-3-methylmalelc anhydride, Rozema et al. were able to retain overall water solubility and réversible Inhibition of membrane activity whlle Incorporating effective In vivo hépatocyte cell targeting. As before, the polynucleotide was covalently linked to the transfection polymer. Covalent attachment of the polynucleotide to the transfection polymer was maintained to ensure co-delivery of the polynucleotide with the transfection polymer to the target cell during in vivo administration by preventing dissociation of the polynucleotide from the transfection polymer. Co-delivery of the polynucleotide and transfection poiymer was requlred because the transfection polymer provided for transport of the polynucleotide across a cell membrane, either from outslde the cell or from inside an endocytic compartment, to the cell cytoplasm. U.S. Patent Publication 20080152661 demonstrated highly efficient delivery of polynucleotides, specifically RNAi oligonucleotides, to liver cells in vivo using this new Improved physiologlcally responsive polyconjugate.

However, covalent attachment of the nucleic acid to the polyamine carries inhérent limitations. Modification of the transfection polymers, to attach both the nucleic acid and the masking agents Is complicated by charge Interactions. Attachment of a negatively charged nucleic acid to a posîtively charged polymer is prône to aggregation thereby limiting the concentration of the mixture. Aggregation can be overcome by the presence of an excess of the polycation or polyanion. However, this solution limits the ratios in which the nucleic acid and the polymer may be formulated. Also, attachment of the negatively charged nucleic acid onto the unmodified cationic polymer causes condensation and aggregation of the complex and Inhibits polymer modification. Modification of the polymer, forming a négative polymer, impairs attachment of the nucleic acid.

SUMMARY OF THE INVENTION

In a preferred embodiment, the invention features a composition for delivering an RNA interférence polynucleotide to a liver cell in vivo comprising: an asialogiycoprotein receptor (ASGPr)-targeted reversibly masked membrane active poiyamine (delivery polymer) and an RNA interférence polynucleotide conjugated to a hydrophobie group containing at least 20 carbon atoms (RNA-conjugate). The delivery polymer and the siRNA-conjugate are syntheslzed separately and may be supplied in separate containers or a single container. The RNA interférence polynucleotide is not conjugated to the polymer.

In a preferred embodiment, the invention features a composition for delivering an RNA interférence poiynucieotide to a liver cell in vivo comprising: an ASGPr-targeted reversibly masked membrane active poiyamine (delivery poiymer) and an RNA interférence poiynucieotide conjugated to a trivalent galactosamine (RNA conjugate). The delivery poiymer and the siRNA-conjugate are synthestzed separateiy and may be supplied in separate containers or a single container. The RNA interférence poiynucieotide is not conjugated to the poiymer.

in a one embodiment, the membrane active poiyamine comprises: an amphipathic poiymer formed by random poiymerizatlon of amine-containtng monomers and lower hydrophobie groupcontalning monomers. The amine-containing monomers contain pendant amine groups selected from the group consisting of: primary amine and secondary amine, The lower hydrophobie monomers contain pendent hydrophobie groups having 1-6 carbon atoms. The ratio of amine groups to hydrophobie groups is selected to form a water solubie poiymer with membrane disruptive activity, preferably £1 amine monomer per hydrophobie monomer. In one embodiment the poiymer wili hâve 60-80% amine monomers. Hydrophobie groups may be selected from the group consisting of: alkyl group, alkenyl group, alkynyl group, aryl group, aralkyl group, aralkenyl group, and aralkynyl group, each of which may be linear, branched, or cyciic. Hydrophobie groups are preferably hydrocarbons, containing only carbon and hydrogen atoms. However, substitutions or heteroatoms which maintaln hydrophobicity, and Inciude, for exemple fluorine, may be permitted. Particularly suitabie membrane active poiyamines comprise poly(vlnyl ether) random copolymers or poly(acrylate) random copolymers.

In a one embodiment, the membrane active poiyamine comprises: an amphipathic poiymer formed by random polymerization of amine-containing monomers, lower hydrophobie monomers, and higher hydrophobie monomers. The amine-containing monomers contain pendant amine groups selected from the group consisting of: primary amine and secondary amine. The lower hydrophobie monomers contain pendent hydrophobie groups having 1-6 carbon atoms. The higher hydrophobie monomers contain pendent hydrophobie groups having 12-36 or more carbon atoms. The ratio of amine groups to hydrophobie groups is selected to form a water solubie poiymer with membrane disruptive activity, preferably £1 amine monomer per hydrophobie monomer. In one embodiment the poiymer wili hâve 60-80% amine monomers. Hydrophobie groups may be selected from the group consisting of: alkyi group, alkenyl group, alkynyl group, aryl group, aralkyl group, aralkenyl group, and aralkynyl group, each of which may be linear, branched, or cyciic, sterol, steroid, and sterold dérivative. Hydrophobie groups are preferably hydrocarbons, containing only carbon and hydrogen atoms. However, substitutions or heteroatoms which maintain hydrophobicity, and include, for example fluorine, may be permitted. Particularfy suitable membrane active polyamines comprise poly(vinyl ether) random terpolymers or poly(acryiate) random terpolymers.

In a preferred embodiment, a reversibly masked membrane active polyamine comprises a membrane active polyamine of the invention reversibly modified by reaction of amines on the polymer with masking agents. An amine is reversibly modified if cleavage of the modifying group restores the amine. Réversible modification of the membrane active polyamine reversibly înhîbits membrane activity of the membrane active polyamine. Preferably, a masking agent also provides targeting function and/or sérum interaction avoidance function. Modification of polymer amine with the masking agent also preferably neutralizes the charge of the amine. A preferred masking agent comprises a galactosamine or galactosamine dérivative or a polyethylene glycol having a disubstituted maleic anhydride amine-reactive group. Reaction of the anhydride with an amine reversibly modifies the amine to form a maieamate or maleamic acid. In the masked state, the reversibly masked membrane active polyamine does not exhibit membrane disruptive activity. Réversible modification of more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, or more than 80%, of the amines on the polyamine with masking agents may be required to inhibit membrane activity and provide cell targeting function, i.e. form a reversibly masked membrane active polymer. Membrane activity Inhibition and/or in vivo targeting of the described membrane active polyamines requires modification of >50% of the polymer amines.

A preferred masking agent comprises a neutral hydrophilic substituted alkylmaleic anhydride:

O

wherein R1 comprises a targeting moiety or a steric stabilizer. An example of a substituted alkylmaleic anhydride constats of a 2-propionic-3-alkyimaleic anhydride dérivative. A neutral hydrophilic 2-proplonlc-3-alkyimaleic anhydride dérivative is formed by attachment of a neutral hydrophilic group to a 2-propionlc-3-alkylmaleic anhydride through the 2-propionic-3-alkylmaleic anhydride γ-carboxyl group. In one embodiment, the alkyt group consists of a methyl group.

A preferred masking agent provides targeting function through affinity for cell surface receptors, i.e. the masking agent contains a ligand for a cell surface receptor. Preferred masking agents contain saccharides having affinity for the ASGPr, including but not limited to: galactose, NAcetyl-galactosamine and galactose dérivatives. Galactose dérivatives having affinity for the ASGPr are well known in the art. An essential feature of the reversibly modified membrane active polyamine Is that at least some, and as many as ail, of the masking agents attached to a polymer provide cell targeting fonction. Another preferred masking agent provides Improved bio-distribution through inhibition of non-specific interactions between the reversibly modified polymer and sérum components or ποη-target cells and by reducing aggregation of the polymer. Preferred masking agents having steric stabilizer fonction inciude, but not limited to, polyethylene glycols. In one embodiment, a combination of targeting and steric stabilizer masking agents are used.

The RNAi polynucleotide conjugate and delivery polymer are administered to a mammal in pharmaceutically acceptable carriers or diluents. In one embodiment, the delivery polymer and the RNAi polynucleotide conjugate may be combined in a solution prior to administration to the mammal. In another embodiment, the delivery polymer and the RNAi polynucleotide conjugate may be co-adminlstered to the mammal in separate solutions. In yet another embodiment, the delivery polymer and the RNAi polynucleotide conjugate may be administered to the mammal sequentially. For sequential administration, the delivery polymer may be administered prior to administration of the RNAI polynucleotide conjugate. Alternative^, for sequential administration, the RNAi polynucleotide conjugate may be administered prior to administration of the delivery polymer.

Further objects, features, and advantages of the invention will be apparent from the following detailed description when taken in conjonction with the accompanying drawings.

Brief Description of the Figures

FIG. 1. Reaction scheme for polymerization of amphipathic poly(vinyl ether) random terpoiymers.

FIG. 2. Graph illustrating the effect of siRNA-cho lesterai conjugate dose on gene knockdown.

FIG. 3. Graph illustrating the effect of hydrophobe size on siRNA-hydrophobe conjugate targeting to liver.

FIG. 4. Graph Illustrating the effect of siRNA-hydrophobe conjugate dose on gene knockdown for several hydrophobie groups.

FIG. 5. Graph illustrating the effect of delivery polymer dose on siRNA-hydrophobe conjugate delivery to liver.

FIG. 6. Llnkage of GaiNAc Cluster to RNA

DETAtLEO DESCRIPTION OF THE INVENTION

Described herein is an improved method for delivering RNA interférence (RNAi) polynucleotides, 5 to iiver cells in a mammal in vivo. The method also provides for improved methods of production of RNAi polynucieotide delivery vehicles. Previously, in vivo delivery of polynucleotides required physical association of the polynucieotide with the delivery vehicle. The polynucieotide was either electrostatically associated with a delivery vehicle, as in poiycation/nucleic acid complexes, encapsulated by the delivery vehicle, as in liposomes and 10 stable nucleic acid-lipid particles (SNALPs), or covalently linked to a delivery vehicie, as in

Dynamic PolyConjugates (Rozema et al. 2007). Surprisingiy, we hâve found that by using approprlate RNAI polynucieotide conjugate molécules and appropriately targeted delivery polymers, the RNAi polynucieotide can be separated from the delivery polymer and still achieve efficient hépatocyte delivery of the polynucieotide.

The ability to separate the polynucieotide from the delivery polymer provides advantages In formulation, synthesis, and manufacturing.

By removing the requirement that the polynucieotide and poiymer are associated, either by covalent linkage or by charge-charge interaction, the concentration of the polymers and polynucleotides and the ratio between them Is limited only by the solubility of the 20 components rather than the solubiiity of the associated complex or ability to manufacture the complex. Increased solubility permits increased polynucieotide or delivery polymer concentration and therefore increased dosage.

The polynucieotide and delivery polymer may be mixed at anytime prior to administration, or even administered separately. Thus, séparation allows the 25 components to be stored separately, either in solution or dry.

Smaller, more stable formulation is possible compared to the larger, inherently unstable non-covalent delivery Systems.

Manufacture of the masked delivery polymer Is easier In the absence of a covalently attached negatively charged polynucieotide or the need to covalently attach a negatively 30 charged polynucieotide.

Manufacture Is simplified and requires fewer steps in absence of physical association of the polynucieotide with the delivery polymer.

Improvements In targeting of the siRNA and polymer are observed.

The Invention Indudes conjugate delivery Systems of the general structure:

(M¹-L)x-P-(L-M²)y Plus N-T, wherein N is a RNAi polynucleotide, T ls a polynucleotide targeting moiety (either a hydrophobie group having 20 or more carbon atoms or a galactose cluster), P ls a membrane active polyamine, and masking agent M¹ contains a targeting moiety, a galactose or galactose dérivative having affinity for the aslaloglycoproteln receptor, covalently linked to P via a physiologically réversible linkage L, such as a maleamate linkage. Cleavage of L restores an unmodified amine on polyamine P. Masking agent M² is optional. If présent, M² is a hydrophilic steric stabilizer covalently linked to P via a physiologically réversible linkage L, such as a maleamate linkage. x and y are each integers. In its unmodified state, P is a membrane active polyamine. Delivery polymer (M¹-L)_x-P-(L-M²)y is not membrane active. Réversible modification of P amines, by attachment of M¹ and optionally M², reversibly inhibits or inactivâtes membrane activity of P and reduces the net positive charge of P. Sufficient masking agents are attached to P to inhibit membrane activity of the polymer. x + y has a value greater than 50%, more preferably greater than 60%, and more preferably greater than 70% of the amines on polyamine P, as determined by the quantity of amines on P in the absence of any masking agents. Upon cieavage of réversible linkages L, unmodified amines are restored thereby reverting P to its unmodified, membrane active state, The réversible bond of réversible linkage L is chosen such that cleavage occurs in a desired physiological condition, such as that présent In a desired tissue, organ, or sub-ceilular location. A preferred réversible linkage ls a pH labile linkage. (M-L^P-fL-M²^, an ASGPr-targeted reversibly masked membrane active polymer (masked polymer), and T-N, a polynucleotide-conjugate, are synthesized or manufactured separately, Neither T nor N are covalently linked directly or indirectiy to P, L, M¹ or M². Electrostatic or hydrophobie association of the polynucleotide or the polynucleotideconjugate with the masked or unmasked polymer is not requlred for in vivo liver delivery of the polynucleotide. The masked polymer and the polynucleotide conjugate can be supplied in the same container or in separate containers. They may be combined prior to administration, coadministered, or administered sequentially.

Polymer The polymers of the invention are amphipathic membrane active poiyamines. A polymer is a molécule built up by répétitive bonding together of smalier units called monomers. A polymer can be a homopolymer in which a single monomer is used or a poiymer can be copolymer or heteropolymer in which two or more different monomers are used, The main chain of a polymer is composed of the atoms whose bonds are required for propagation of polymer length. A slde chain of a polymer is composed of the atoms whose bonds are not requlred for propagation of polymer length.

More specifïcally, the polymers of the invention are amphîpathic membrane active random copolymers. The monomers in random copolymers do not hâve a defined or arrangement along the main chain, and are written, for example, as: -A_x-B_y- or -A_x-B_y-C_z-. The general compositions of such polymers are reflective of the ratio of input monomers. However, the exact ratio of one monomer to another may differ between chains. The distribution of monomers may also differ along the length of a single polymer. Also, the chemlcal properties of a monomer may affect its rate of incorporation Into a random copolymer and its distribution within the poiymer. While the ratio of monomers in a random polymer Is dépendent on the input ratio of monomer, the input ratio may not match exactly the ratio of Incorporated monomers.

Amphîpathic

Amphîpathic, or amphiphilic, polymers are well known and recognized in the art and hâve both hydrophilic (polar, water-soluble) and hydrophobie (non-polar, lipophilie, water-insoluble) groups or parts.

Hydrophilic groups indicate in qualitative terms that the chemical moiety is water-preferring. Typically, such chemical groups are water soluble, and are hydrogen bond donors or acceptors with water. A hydrophilic group can be charged or uncharged. Charged groups can be positively charged (anlonic) or negatively charged (cationic) or both (zwitterionic). Examples of hydrophilic groups include the following chemical moieties: carbohydrates, polyoxyethyiene, certain peptides, oligonucleotides, amines, amides, alkoxy amldes, carboxylïc acids, sulfurs, and hydroxyls.

Hydrophobie groups indicate in qualitative terms that the chemical moiety is water-avoiding. Typically, such chemical groups are not water soluble, and tend not to form hydrogen bonds. Lipophilie groups dissolve in fats, oils, lipids, and non-polar solvents and hâve little to no capacity to form hydrogen bonds. Hydrocarbons containing two (2) or more carbon atoms, certain substituted hydrocarbons, cholestérol, and cholestérol dérivatives are exampies of hydrophobie groups and compounds.

As used herein, with respect to amphipathlc polymers, a part is defined as a molécule derived when one covalent bond is broken and replaced by hydrogen. For example, in butyl amine, a breakage between the carbon and nitrogen bonds, and replacement with hydrogens, results In ammonla (hydrophilic) and butane (hydrophobie). If 1,4-diaminobutane is cleaved at nitrogencarbon bonds, and replaced with hydrogens, the resulting molécules are again ammonia (2*) and butane. However, 1,4,-diaminobutane Is not considered amphîpathic because formation of the hydrophobie part requires breakage of two bonds.

As used herein, a surface active polymer lowers the surface tension of water and/or the Interfacial tension with other phases, and, accordingly, is positively adsorbed at the liquid/vapor Interface. The property of surface activity is usually due to the fact that the molécules of the substance are amphipathlc or amphiphilic.

Membrane Active

As used herein, membrane active polymers are surface active, amphipathic polymers that are able to Induce one or more of the following effects upon a biological membrane: an alteration or disruption of the membrane that allows non-membrane permeable molécules to enter a cell or cross the membrane, pore formation in the membrane, fission of membranes, or disruption or dissolving of the membrane. As used herein, a membrane, or cell membrane, comprises a iipid bilayer. The alteration or disruption of the membrane can be functionaliy defined by the polymer's activity in at least one the following assays: red blood cell iysis (hemoiysis), liposome ieakage, liposome fusion, cell fusion, cell Iysis, and endosomal release. Membrane active polymers that can cause iysis of cell membranes are also termed membrane lytic polymers. Polymers that preferentially cause disruption of endosomes or lysosomes over plasma membrane are considered endosomolytic. The effect of membrane active polymers on a cell membrane may be transient. Membrane active polymers possess affinity for the membrane and cause a dénaturation or deformation of bilayer structures. Membrane active polymers may be synthetic or non-natural amphipathic polymers.

As used herein, membrane active polymers are distinct from a class of polymers termed cell penetrating peptides or polymers represented by compounds such as the arginine-rich peptide derived from the HIV TAT protein, the antennapedia peptide, VP22 peptide, transportan, arginine-rich artificlai peptides, small guanidinium-rich artificlai polymers and the iike, While cell penetrating compounds appear to transport some molécules across a membrane, from one side of a iipid bilayer to other slde of the iipid bilayer, apparently without requiring endocytosis and without disturbing the integrity of the membrane, their mechanism is not understood.

Delivery of a polynucleotide to a cell Is mediated by the membrane active polymer disrupting or destabilizing the plasma membrane or an internai vesicle membrane (such as an endosome or lysosome), including forming a pore In the membrane, or disrupting endosomal or lysosomal vesicles thereby permitting release of the contents of the vesicle into the cell cytoplasm.

Endosomolytic

Endosomolytic polymers are polymers that, in response to a change in pH, are able to cause disruption or Iysis of an endosome or provide for release of a normally cell membrane imperméable compound, such as a polynucleotide or protein, from a cellular Internai membrane-enclosed veslcle, such as an endosome or lysosome. Endosomolytic polymers undergo a shift in their physlco-chemical properties over a physiologically relevant pH range (usually pH 5.5 - 8). This shift can be a change In the polymefs solubility or ability to Internet with other compounds or membranes as a resuit in a shift in charge, hydrophobicity, or hydrophilicity. Exemplary endosomolytic polymers hâve pH-iabïle groups or bonds. A reversibly masked membrane active polymer, wherein the masking agents are attached to the polymer via pH labile bonds, can therefore be considered to be an endosomolytic polymer.

Amphipathic Membrane Active Random Copolymers Amphlpathic membrane active polyamines of the invention comprise: amphipathic membrane active polyamines (random heteropoiymers).

For copolymers of the invention, the two or more monomeric species consist minimally of: a monomer contalning a pendant primary or secondary amine group and a monomer containing a pendant hydrophobie pendent group. In a more preferred embodiment, the two monomer species consist minimally of: a monomer containing a pendant primary or secondary amine group and a monomer containing a pendant lower hydrophobie pendent group. As used herein, a pendant group is a group composed of the atoms linked to a polymer but whose bonds are not requlred for propagation of polymer iength, I.e., neither the atoms nor bonds of a pendant group are part of the main chain or backbone of a polymer to which the group is attached.

Amphipathic membrane active polyamine copolymers of the invention are the product of copolymerization of two or more monomer species. In one embodiment, amphipathic membrane active heteropoiymers of the invention hâve the general structure:

-(A).-(B)_bwherein, A contalns a pendent primary or secondary amine functional group and B contains a lower hydrophobie pendant group (containing 2 to about 6 carbon atoms). a and b are integers >0. To aid In synthesis, protected amine containing monomers, such as phthalimido-protected or BOC-protected amine monomers may be used during polymerization. The amine protecting groups are removed after polymerization to yield amines. The Incorporation of monomers, up to 10%, containing pendant medium or higher hydrophobie groups (7 or more carbon atoms) Is permissible. The Incorporation of additional monomeric species in minor amounts (<5%) Is also permissible. For example, polymers may also hâve additional reactive group-containing monomers. Reactive group-containing monomers may be used to attach components to the polymer following synthesis of the polymer. A monomer can hâve a reactive group that does not participate in the polymerization reaction. A monomer can also hâve a reactive group that Is protected. The protection group prevents reaction of the reactive group during polymerization. After polymerization, the protection group is removed.

In another embodiment a terpolymer, a poiymer having at least three different monomeric species, is used as the delivery poiymer. For terpolymers of the invention, the three monomeric species consist minimally of: a monomer containing a pendant primary or secondary amine group, a monomer containing a fïrst pendant hydrophobie group, and a monomer containing a second pendant hydrophobie group wherein the first and second hydrophobie pendent groups are different. In a more preferred embodiment, the three or more monomers species consist minimally of: a monomer containing a primary or secondary amine group, a monomer containing a pendant iower hydrophobie group, and a monomer containing a pendant medium or higher hydrophobie group.

In one embodiment, amphipathic membrane active terpolymers of the invention hâve the general structure:

-(A).-(B)_b-(C)_cwherein, A contains a pendent primary or secondary amine functional group, B contains a pendant iower hydrophobie group (containing 2 to about 6 carbon atoms), and C contains a pendant higher hydrophobie group (containing 12 or more carbon atoms). a, b, and c are integers >0. To aid in synthesis, protected amine-containing monomers, such as phthalimidoprotected or BOC-protected amine monomers may be used during polymerization. The amine protecting groups are removed after polymerization to yield amines. The Incorporation of additlonal monomeric species in minor amounts (<5%) is possible. For example, polymers may also hâve additionai hydrophobie monomers or reactive group-containing monomers. Reactive group-containing monomers may be used to attach components to the poiymer foilowing synthesis of the poiymer. A monomer can hâve a reactive group that does not participate In the polymerization reaction. A monomer can also hâve a reactive group that Is protected. The protection group prevents reaction of the reactive group during polymerization. After polymerization, the protection group is removed.

Hydrophobie groups are preferabiy hydrocarbons, containing only carbon and hydrogen atoms. However, non-polar substitutions or non-polar heteroatoms whlch maintain hydrophoblcity, and include, for example fluorine, may be permitted. The term Includes aliphatic groups, aromatic groups, acyl groups, alkyl groups, alkenyi groups, alkynyl groups, aryl groups, aralkyl groups, araikenyi groups, and aralkynyi groups, each of which may be linear, branched, or cyciic. The term hydrophobie group also includes: sterols, steroids, cholestérol, and steroid and cholestérol dérivatives. As used herein, Iower hydrophobie monomers or groups comprise hydrophobie 12 groups having two (2) to six (6) carbon atoms. As used herein, medium hydrophobie monomers or groups comprise hydrophobie groups having seven (7) to eleven (11) carbon atoms. As used herein, higher hydrophobie monomers or groups comprise hydrophobie groups having twelve (12) to thlrty-sic (36) or more carbon atoms.

The biophysical properties of the amphipathîc polymère are determined by the classes of monomer specles polymerized, the ratio at which they are incorporated Into the polymer, and the size of the polymer. Different polymère can be made by aitering the feed ratio of monomère in the polymerization reaction or aitering the groups used to modify a polymer backbone. While the Incorporated ratio of monomère In a polymer can be the same as the feed ratio of monomers, the ratios can be different. Whetherthe monomers are incorporated at the feed ratio or at a different ratio, It is possible to alter the feed ratio of monomère to achieve a desired monomer Incorporation ratio.

The ratio of amine groups to hydrophobie groups Is selected to form a water soluble polymer with membrane disruptive activlty. Preferred membrane active polymère of the invention are water soluble at £1 mg/ml, £5 mg/ml, £10 mg/ml, £15 mg/ml, £20 mg/ml, £25 mg/ml, and £30 mg/ml. Preferred membrane active polymère of the invention are surface active. Membrane active polymère of the invention are preferably In the size range of about 3 kDa to about 300 kDa. Because the polymère are amphipathîc, they self-associate In aqueous solution, with a critical association concentration S1 mg/ml.

In one embodiment, the monomer Incorporation ratio for the membrane active polyamine copolymers is about 4-8 amine monomers : 3-5 lower hydrophobie monomers. In another embodiment, the monomer incorporation ratio for the membrane active polyamlnes Is about 5.4-7.5 amine monomère : 3-3.5 lower hydrophobie monomère. In another embodiment, the monomer Incorporation ratio for the membrane active polyamines Is about 2 amine monomers to about 1 lower hydrophobie monomers. In one embodiment the hydrophobie groups of the hydrophobie monomers consist of alkyl groups.

In one embodiment, the monomer incorporation ratio for the membrane active polyamine terpolymere is about 4-8 amine monomère : 3-5 lower hydrophobie monomers : 1 higher hydrophobie monomer. In another embodiment, the monomer incorporation ratio for the membrane active polyamines Is about 5.4-7.5 amine monomère : 3-3.5 lower hydrophobie monomers : 1 higher hydrophobie monomère. In another embodiment, the monomer incorporation ratio for the membrane active polyamines is about 6 amine monomers to about 3 lower hydrophobie monomers to about 1 higher hydrophobie monomer. In one embodiment the hydrophobie groups of the hydrophobie monomers consist of alky! groupe.

In one embodiment, the amine/lower hydrophobie group copolymers are synthesized using monomers at a feed ratio of about 4-8 amine monomer : about 3-5 lower alkyl monomer. In 5 another embodiment, the amine/lower hydrophobie group copolymers can be synthesized using monomers at a feed ratio of about 15 amine monomer : 4 lower hydrophobie group monomer.

In one embodiment, the amine/lower hydrophobie group/higher hydrophobie group terpolymers are synthesized using monomers at a feed ratio of about 4-8 amine monomer : about 3-5 lower alky! monomer : 1 higher alkyl monomer. In another embodiment, the amine/lower hydrophobie 10 group/higher hydrophobie group terpolymers can be synthesized using monomers at a feed ratio of about 15 amine monomer : 4 lower hydrophobie group monomer : 1 higher hydrophobie group monomer.

In one embodiment, particulariy suitable membrane active polyamines comprise copolymers having amine containing monomers, butyl-containing monomers and higher hydrophobie group15 containing monomers wherein the higher hydrophobie group contains 12-18 carbon atoms.

Particulariy suitable membrane active polyamines comprise poly(vinyl ether) random terpolymers or poly(acrylate) random terpolymers.

In another embodiment, particulariy suitable membrane active polyamines comprise copolymers having amine containing monomers, lower hydrophobie group-containing 20 monomers. Particulariy suitable membrane active polyamines comprise poly(vlnyl ether) random copolymers or poly(acrylate) random copolymers.

Particulariy suitable membrane active polyamines comprise copolymers having amine containing monomers and butyl-containing monomers. Particulariy suitable membrane active polyamines comprise poly(vinyl ether) random copolymers or poly(acrylate) random copolymers.

Biodégradable Polymère

A polymer may hâve one or more cleavable bonds. If the cleavable bonds are naturally cleaved under physiological conditions or celiular physiological conditions, the polymer is biodégradable. The biodégradable bond may either be in the main-chain or in a side chain. If the cleavable bond occurs in the main chain, cleavage of the bond results in a decrease in polymer length 30 and the formation of two moiecules. If the cleavable bond occurs in the side chain, then cleavage of the bond results In loss of side chain atoms from the polymer. For membrane active polymère, biodégradation of the polymer will resuit In decreased membrane activity of the polymer. As used herein, the term biodégradable means that the polymer will dégradé over time by the action of enzymes, by hydrolytic action and/or by other similar mechanisms in the body. Biodégradable bonds are those bonds which are cleaved by biologlcal processes and include, but are not limited to: esters, phosphodiesters, certain peptide bonds and combinations thereof. Esters undergo hydrolysis and are also catalyticaily cleaved by esterases. Phosphodiesters are cleaved by nucleases. Peptide bonds are cleaved by peptidases. In particuiar, the polymer backbone is degraded or cleaved, or slde chains (pendent groups) are degraded or cleaved, from the polymer. Biodégradable bonds In the biodégradable polymers may be cleaved, under physiological conditions with a half iife of less than 45 min, more than 45 minutes, more than 2 hours, more than 8 hours, more than 24 hours, or more than 48 hours. While biodégradable polymers are useful for In vivo delivery, the polymer must be sufficientiy stable to form a sufficiently sized polymer In aqueous solution. Also, the rate of cleavage of a biodégradable bond must be slower than the labile bond used to attach a masking agent to the polymer. In a preferred embodiment, dégradation of a biodégradable polymer occurs at a slower rate than cleavage of the masking agents.

Masking

The delivery polymers of the invention comprise reversibly modified amphipathic membrane active polyamines wherein réversible modification inhibits membrane activity, neutralizes the polyamine to reduce positive charge and form a near neutral charge polymer, provides cell-type spécifie targeting, and inhibits non-specific interactions of the polymer. The poiyamine is reversibly modified through réversible modification of amines on the polyamine.

The membrane active polyamines of the invention are capable of disrupting plasma membranes or iysosomal/endocytic membranes. This membrane activity Is an essential feature for celiular delivery of the polynucleotide. Membrane activity, however, leads to toxicity when the polymer is administered in vivo. Polyamines also Internet readily with many anionic components in vivo, leading to undeslred bio-distribution. Therefore, réversible masking of membrane activity of the polyamine Is necessary for in vivo use. This masking Is accomplished through réversible attachment of masking agents to the membrane active polyamine to form a reversibly masked membrane active polymer, Le. a delivery polymer. In addition to inhibiting membrane activity, the masking agents shield the polymer from non-specific interactions, reduce sérum interactions, increase circulation time, and provide cell-specific interactions, i.e. targeting.

It is an essential feature of the masking agents that, in aggregate, they inhibit membrane activity of the polymer, shield the polymer from non-specific interactions (reduce sérum interactions, increase circulation time), and provide in vivo hépatocyte targeting. The membrane active polyamine Is membrane active in the unmodified (unmasked) state and not membrane active (inactivated) in the modified (masked) state. A sufficient number of masking agents are linked to the polymer to achleve the desired ievel of Inactivation. The deslred level of modification of a polymer by attachment of masking agent(s) is readily determined using appropriate polymer activity assays. For example, if the polymer possesses membrane activity in a given assay, a sufficient levei of masking agent is iinked to the polymer to achieve the desired level of inhibition of membrane activity in that assay. Masking requires modification of £50%, £60%, £70%, or £60% of the amine groups on the polymer, as determined by the quantity of amines on the polymer In the absence of any masking agents, it Is also a preferred characteristic of masking agents that their attachment to the polymer reduces positive charge of the polymer, thus forming a more neutral delivery polymer. It Is désirable that the masked polymer retain aqueous solubility.

As used herein, a membrane active polyamine is masked if the modified polymer does not exhibit membrane activity and exhibits cell-specific (i.e. hépatocyte) targeting in vivo. A membrane active polyamine is reversibly masked If cleavage of bonds linking the masking agents to the polymer results In restoration of amines on the polymer thereby restorlng membrane activity.

it is another essentiai feature that the masking agents are covalently bound to the membrane active polyamine through physlologically réversible bonds. By using physiologically réversible linkages or bonds, the masking agents can be cleaved from the polymer In vivo, thereby unmasking the polymer and restoring activity of the unmasked polymer. By choostng an appropriate réversible linkage, it is possible to form a conjugale that restores activity of the membrane active polymer after it has been delivered or targeted to a desired cell type or cellular location. Reverslbility of the linkages provides for sélective activation of the membrane active polymer. Réversible covalent linkages contain réversible or labile bonds which may be selected from the group comprlsing: physlologically labile bonds, cellular physiologically labile bonds, pH labile bonds, very pH labile bonds, and extremeiy pH labile bonds.

As used herein, a masking agent comprises a compound having an ASGPr targeting moiety or a steric stabilizer and an amlne-reactive group wherein reaction of the amine-reactive group with an amine on a polymer results in linkage of the ASGPr targeting moiety or steric stabilizer to the polymer via a physiologically labile covalent bond. An ASGPr targeting moiety is a group, typically a saccharide, having affinity for the asialoglycoprotein receptor. A preferred steric stabilizer Is a polyethyiene glycoi (PEG). Preferred masking agents of the Invention are able to modify the polymer (form a réversible bond with the polymer) in aqueous solution. A preferred 16 amine-reactive group comprises a disubstituted maleic anhydride. A preferred masking agent Is represented by the structure:

wherein in which R¹ is an alkyl group such as a methyl (-CHj) group, ethyl (-CH2CH3) group, or propyl (-CH₂CH₂CH₃) group (to form a substituted alkylmaleic anhydride), and R² comprises an asialoglycoprotein receptor (ASGPr) targeting molety or a steric stabilizer.

The membrane active polyamine can be conjugated to masking agents In the presence of an excess of masking agents. The excess masking agent may be removed from the conjugated delivery polymer prior to administration of the delivery polymer.

Steric Stabilizer

As used herein, a steric stabilizer is a non-lonic hydrophilic polymer (either natural, synthetic, or non-natural) that prevents or Inhibits Intramolecular or Intermolecular Interactions of a polymer to which it Is attached relative to the polymer containing no steric stabilizer. A steric stabilizer hinders a polymer to which it Is attached from engaging in electrostatic Interactions. Electrostatic interaction Is the non-covalent association of two or more substances due to attractive forces between positive and négative charges. Steric stabliizers can Inhibit Interaction with blood components and therefore opsonization, phagocytosls, and uptake by the réticuloendothélial System. Steric stabliizers can thus Increase circulation time of molécules to which they are attached. Steric stabilîzers can also Inhibit aggregation of a polymer. A preferred steric stabilizer Is a polyethylene glycol (PEG) or PEG dérivative. As used herein, a preferred PEG can hâve about 1-500 ethylene glycol monomers, 2-20 ethylene glycol monomers, 5-15 ethylene glycol monomers, or about 10 ethylene glycol monomers. As used herein, a preferred PEG can also hâve a molecular welght average of about 85-20,000 Daltons (Da), about 2001000 Da, about 200-750 Da, or about 550 Da. As used herein, steric stabliizers prevent or inhibit intramolecular or intermolecular Interactions of a polymer to which It Is attached relative to the polymer containing no steric stabilizer In aqueous solution.

ASGPr Targeting Moiety

Targeting moieties or groupe enhance the pharmacokinetic or biodistribution properties of a conjugate to which they are attached to Improve cell-specific distribution and cell-speclfic uptake of the conjugate. Galactose and galactose dérivâtes hâve been used to target molécules to hépatocytes in vivo through their binding to the asîaloglycoproteîn receptor (ASGPr) expressed on the surface of hépatocytes. As used hereln, a ASGPr targeting moiety comprises a galactose and galactose dérivative having affinity for the ASGPr equal to or greater than that of galactose. Binding of galactose targeting moieties to the ASGPr(s) facilitâtes cellspécifie targeting of the delivery polymer to hépatocytes and endocytosis of the delivery polymer into hépatocytes.

ASGPr targeting moieties may be selected from the group comprising: lactose, galactose, Nacetyl galactosamine (GalNAc), galactosamine, N-formylgalactosamine, N-acetyl-galactosamine, N-propionylgalactosamlne, N-n-butanoylgalactosamine, and N-iso-butanoyl-galactosamine (lobst, S.T. and Drickamer, K. J.B.C. 1996, 271, 6686). ASGPr targeting moieties can be monomeric (e.g., having a single galactosamine) or multimeric (e.g., having multiple galactosamines).

In some embodiments, the galactose targeting moiety is linked to the amine-reactive group through a PEG linker as illustrated by the structure:

wherein n is an integer between 1 and 19.

In one embodiment, the membrane active polyamine is reversibly masked by attachment of ASGPr targeting moiety masking agents to £50%, £60%, £70%, or £80% of amines on the polyamine. In another embodiment, the membrane active polyamine is reversibly masked by attachment of ASGPr targeting moiety masking agents and PEG masking agents to £50%, £60%, £70%, or £80% of amines on the polyamine. In another embodiment, the ASGPr targeting moiety masking agents comprise an ASGPr targeting moiety linked to an aminereactive group via a PEG linker. For membrane active polyamine masking with both ASGPr targeting moiety masking agents and PEG masking agents, a ratio of PEG to ASGPr targeting moiety is about 0-4:1, more preferabiy about 0.5-2:1. In another embodiment, there are about 1.3-2 PEG masking agents to about 1 galactose dérivative masking agent

Surface Charge

Zêta potential is a physical property which is exhibited by a particie in suspension and is closely related to surface charge. In aqueous media, the pH of the sample is one of the most important factors that affects zêta potential. When charge Is based upon protonation/deprotonation of bases/aclds, the charge is dépendent on pH. Therefore, a zêta potential value must include the solution conditions, especially pH, to be meaningful. For typical partlcles, the magnitude of the zêta potential gives an indication of the potential stability of the colloïdal System. If ail the particles in suspension hâve a large négative or positive zêta potential, they will tend to repel each other and there will be no tendency for the particles to corne together. However, if the particles hâve low zêta potential values, there will be no force to prevent the particles coming together and flocculating. The general dividing line between stable and unstabte suspensions for typical particles is generally taken at either +30 or -30 mV. Particles with zêta potentials more positive than +30 mV or more négative than -30 mV are normally considered stable. Delivery polymers of the described invention exhlbit a zêta potential of 20 mV to -20 mV at physiological sait and pH 8, but are colloidally stable in aqueous solution and do not flocculate.

Positive charge, or zêta potential, of a membrane active polyamine Is reduced by modification with the masking agents. Polymer charge, especially positive charge, can resuit in unwanted interactions with sérum components or non-target cells. Positive surface charge also plays a rôle in membrane activity by enhancing Interaction of the polymer with negatively charged cell membranes. Therefore, delivery polymers with near neutral net charge or zêta potential are preferred for in vivo delivery of polynucleotides. Delivery polymers of the Invention, membrane active polyamines masked by réversible attachment of ASGPr targeting moiety masking agents and steric stabilizer masking agents, hâve an apparent surface charge near neutral and are sérum stable. More specifically, the delivery polymers of the invention hâve a zêta potential, measured at pH 8, between +30 and -30 mV, between +20 and -20 mV, between +10 and -10 mV, or between +5 and -5 mV. At pH 7, the net charge of the conjugate is expected to be more positive than at pH 8. Net charge, or surface charge, is a significant factor for In vivo applications.

Labile Unkage

A linkage or linker Is a connection between two atoms that links one chemlcal group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. For example, a linkage can connect a masking agent to a polymer. Formation of a linkage may connect two separate molécules Into a single molécule or It may connect two atoms in the same molécule. The linkage may be charge neutral or may bear a positive or négative charge. A réversible or labile linkage contains a réversible or labile bond. A linkage may optionally Include a spacer that Increases the distance between the two joined atoms. A spacer may further add flexibility and/or length to the llnkage. Spacers may inciude, but are not be limited to, alkyi groups, alkenyl groups, alkynyl groupe, aryl groups, araikyl groups, aralkenyi groups, aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, 5 nucléotides, and saccharides. Spacer groups are weli known In the art and the preceding list Is not meant to llmit the scope of the Invention.

A réversible or labile bond Is a covalent bond other than a covalent bond to a hydrogen atom that Is capable of belng selectively broken or cleaved under conditions that will not break or cleave other covalent bonds In the same molécule. More specifically, a réversible or labile bond 10 is a covalent bond that Is less stable (thermodynamically) or more rapidly broken (kinetically) under appropriate conditions than other non-labile covalent bonds In the same molécule. Cleavage of a labile bond within a molécule may resuit In the formation of two molécules. For those skilled in the art, cleavage or lability of a bond is generally discussed in terms of half-life (tü) of bond cleavage (the time required for half of the bonds to cleave). Thus, réversible or 15 labile bonds encompass bonds that can be selectively cleaved more rapidly than other bonds a molécule.

Appropriate conditions are determined by the type of labile bond and are well known In organic chemistry. A labile bond can be sensitive to pH, oxidative or reductive conditions or agents, température, sait concentration, the presence of an enzyme (such as esterases, including 20 nucleases, and proteases), or the presence of an added agent. For example, increased or decreased pH Is the appropriate conditions for a pH-labile bond.

The rate at which a labile group will undergo transformation can be controlled by altering the chemlcal constituées of the molécule containing the labile group. For example, addition of particular chemical moletles (e.g., électron acceptors or donors) near the labile group can affect 25 the particular conditions (e.g., pH) under which chemlcal transformation will occur.

As used herein, a physiologically labile bond Is a labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammallan body. Physiologically labile linkage groups are selected such that they undergo a chemical transformation (e.g., cleavage) when présent in certain physiological conditions.

As used herein, a cellular physiologically labile bond Es a labile bond that is cleavable under mammalian Intracellular conditions. Mammalian Intracellular conditions Inciude chemical conditions such as pH, température, oxidative or reductive conditions or agents, and sait concentration found In or analogous to those encountered In mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activity normally présent ln a mammalian cell such as from proteolytic or hydrolytic enzymes. A cellular physiologically labile bond may also be cleaved In response to administration of a pharmaceutically acceptable exogenous agent. Physiologically labile bonds that are cleaved under appropriate conditions with a half life of less than 45 min. are considered very labile. Physiologically labile bonds that are cleaved under appropriate conditions with a half life of less than 15 min are considered extremely labile.

Chemical transformation (cleavage of the labile bond) may be initiated by the addition of a pharmaceutically acceptable agent to the cell or may occur spontaneously when a molécule containing the labile bond reaches an appropriate Intra-and/or extra-cellular environment. For example, a pH labile bond may be cleaved when the molécule enters an acidified endosome. Thus, a pH labile bond may be considered to be an endosomal cleavable bond. Enzyme cleavable bonds may be cleaved when exposed to enzymes such as those présent in an endosome or lysosome or in the cytoplasm. A disulfide bond may be cleaved when the molecuie enters the more reducing environment of the cell cytoplasm. Thus, a disulfide may be considered to be a cytoplasmic cleavable bond.

As used herein, a pH-labile bond is a labile bond that is selectively broken under acldic conditions (pH<7). Such bonds may also be termed endosomally labile bonds, since cell endosomes and lysosomes hâve a pH less than 7. The term pH-labile includes bonds that are pH-labile, very pH-labile, and extremely pH-labile.

Reaction of an anhydride with an amine forms an amide and an acid. For many anhydrides, the reverse reaction (formation of an anhydride and amine) is very slow and energetically unfavorable. However, if the anhydride is a cyclic anhydride, réaction with an amine yields an amide acid, a molecuie in which the amide and the acid are in the same molecuie. The presence of both reactive groups (the amide and the carboxylic acid) in the same molecuie accelerates the reverse reaction. In particular, the product of primary amines with maleic anhydride and maleic anhydride dérivatives, maleamic acids, revert back to amine and anhydride 1*10* to 1*10” times faster than its noncyclic analogues (Kirby 1980).

Reaction of an amine with an anhydride to form an amide and an acid.

_ Q _r

2I

Reaction of an amine with a cyclic anhydride to form an amide acid.

0	q
	.R1		R hX	.R1
	Z	‘OH
r-nh2 + o	t	u +
	XR2	Π	T	R2
0			O

Cleavage of the amide acid to form an amine and an anhydride is pH-dependent and Is greatly accelerated at acidic pH. This pH-dependent reactivity can be exploited to form réversible pHlabile bonds and linkers. Cis-aconitic acid has been used as such a pH-sensitive linker molécule. The γ-carboxylate is fîrst coupled to a molécule. In a second step, either the a or β carboxylate Is coupled to a second molécule to form a pH-sensitive coupling of the two molécules. The half life for cleavage of this linker at pH 5 Is between 8 and 24 h.

Structures of cis-aconitic anhydride and maleic anhydride.

O O H H

aconitic acid maleic anhydride

The pH at whlch cleavage occurs is controlled by the addition of chemical constituents to the labile moiety. The rate of conversion of maleamic acids to amines and maleic anhydrides is strongly dépendent on substitution (R2 and R3) of the maleic anhydride System. When R2 is methyl, the rate of conversion is 50-fold higher than when R2 and R3 are hydrogen. When there are alkyl substitutions at both R2 and R3 (e.g., 2,3-dimethylmaleicanhydride) the rate increase is dramatic: 10,000-fold faster than non-substituted maleic anhydride. The maleamate bond formed from the modification of an amine with 2,3-dimethylmaleic anhydride is cleaved to restore the anhydride and amine with a half-life between 4 and 10 min at pH 5. It is anticipated that if R2 and R3 are groups larger than hydrogen, the rate of amide-acid conversion to amine and anhydride wlll be faster than If R2 and/or R3 are hydrogen.

Very pH-labile bond: A very pH-labile bond has a half-life for cleavage at pH 5 of less than 45 min. The construction of very pH-labile bonds is well-known In the chemical art.

Extremely pH-labile bonds: An extremely pH-labile bond has a half-life for cleavage at pH 5 of less than 15 min. The construction of extremely pH-labile bonds Is well-known in the chemical art.

Disubstituted cyclic anhydrides are particulariy useful for attachaient of masking agents to membrane active polyamines of the invention. They provide physîologically pH-labile linkages, readily modify amines, and restore those amines upon cleavage in the reduced pH found In cellular endosomes and lysosome. Second, the a or β carboxylic acid group created upon reaction with an amine, appears to contribute only about 1 /2O'^h of the expected négative charge to the polymer (Rozema et al. Bioconjugate Chemistry 2003). Thus, modification of the 10 pdyamine with the disubstituted maleic anhydrides effectively neutralizes the positive charge of the polyamine rather than créâtes a polymer with high négative charge. Near neutral polymers are preferred for in vivo delivery.

Step Polymerization

In step polymerization, the polymerization occurs in a stepwise fashion. Polymer growth occurs 15 by reaction between monomers, oligomers, and polymers. No initiator is needed since the same reaction occurs throughout, and there Is no termination step so that the end groups are still reactive. The polymerization rate decreases as the functional groups are consumed.

A polymer can be created using step polymerization by using monomers that hâve two reactive groups (A and B) In the same monomer (heterobifunctional), wherein A comprises a reactive 20 group and B comprises an A-reactive group (a reactive group which forms a covalent bond with A). Polymerization of A-B yieids —{A—B]_n—. Reactive groups A and B can be joined by a covalent bond or a plurality of covalent bonds, thereby forming the polymer monomer. A polymer can also be created using step polymerization by using homobifunctional monomers such that A-A + B-B yieids -{A-A-B-Bjr,-. Generally, these reactions can Involve acylation or 25 alkylation. The two reactive groups of a monomer can be joined by a single covalent bond or a plurality of covalent bonds.

If reactive group A is an amine then B is an amine-reactive group, which can be selected from the group comprising: isothiocyanate, isocyanate, acyl azide, N-hydroxy-succinimide, sulfonyl chloride, aldéhyde (including formaldéhyde and glutaraldehyde), ketone, epoxide, carbonate, 30 imldoester, carboxylate activated with a carbodiimide, alkylphosphate, arylhalides (dîfluorodinitrobenzene), anhydride, acid halide, p-nitrophenyl ester, o-nitrophenyl ester, pentachlorophenyl ester, pentafluoropheny! ester, carbonyl imldazole, carbonyl pyridinium, and carbonyl dimethylaminopyridinium. In other ternis, when reactive group A is an amine then B can be an acylating or alkylating agent or amination agent

If reactive group A is a sulfhydryl (thlol) then B is a thlol-reactive group, which can be selected from the group comprising: iodoacetyl dérivative, maleimide, aziridine dérivative, acryloyl dérivative, fluorobenzene dérivative, and disulfide dérivative (such as a pyridyl disuifide or 5thio-2-nitrobenzoic acid (TNB) dérivative).

If reactive group A is carboxylate then reactive group B is a carboxylate-reactive group, which can be selected from the group comprising: diazoacetate and an amine in which a carbodiimide is used. Other additives may be utilized such as carbonyldiimidazole, dimethylamino pyridine 10 (DMAP), N-hydroxysuccinimide or alcohol using carbodiimide, and DMAP.

If reactive group A Is a hydroxyl then réactivé group B is a hydroxyl-reactive group, which can be selected from the group comprising: epoxide, oxlrane, activated carbamate, activated ester, and alkyl halide.

If reactive group A is an aldéhyde or ketone then reactive group B is an aldéhyde- or ketone15 reactive group, which can be selected from the group comprising: hydrazine, hydrazide dérivative, amine (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBHj), and hydroxyl compound.

A poiymer can be created using step polymerization by using bifunctional monomers and another agent such that A-A plus another agent yields —[A—A]_n—.

If reactive group A is a sulfhydryi (thiol) group then It can be converted to disulfide bonds by oxidizing agents such as iodine (l₂), sodium periodate (NalO₄), or oxygen (O₂). If reactive group A can is an amine, it can be converted to a thiol by reaction with 2-lminothiolate (Traut’s reagent) which then undergoes oxidation and disulfide formation. Disulfide dérivatives (such as a pyridyl disulfide or TNB dérivatives) can also be used to catalyze disulfide bond formation.

Reactive groups A or B in any of the above examples can also be a photoreactive group such as aryl azide (including halogenated aryl azide), diazo, benzophenone, alkyne, or diazirine dérivative.

Reactions of the amine, hydroxyl, sulfhydryl, or carboxylate groups yield chemical bonds that are described as amldes, amidines, disuifides, ethers, esters, enamines, imines, ureas, 30 isothioureas, Isoureas, sulfonamides, carbamates, alkylamine bonds (secondary amines), and carbon-nitrogen single bonds In which the carbon Is boned to a hydroxyl group, thloether, diol, hydrazone, diazo, or sulfone.

Chain Poiymerization In chain-reactlon poiymerization, growth of the polymer occurs by successive addition of monomer units to a limited number of growing chains. The initiation and propagation mechanisms are different, and there is typically a chain-terminating step, Chain poiymerization reactions can be radical, anionlc, or cationic. Monomers for chain poiymerization may be selected from the groups comprislng: vinyl, vinyl ether, acrylate, méthacrylate, acrylamlde, and methacrylamlde groups. Chain poiymerization can also be accomplished by cycle or ring opening poiymerization. Several different types of free radical Initiators can be used including, but not limited to, peroxldes, hydroxy peroxides, and azo compounds such as 2,2'-Azobis(amidinopropane) dihydrochloride (AAP).

A naturaliy occurring polymer is a polymer that can be found in nature. Examples include polynucleotldes, proteins, coiiagen, and polysaccharides (starches, ceilulose, glycosaminoglycans, chitin, agar, agarose). A natural polymer can be Isolated from a biological source or it can be synthetic. A synthetlc polymer Is formulated or manufactured by a chemical process by man and is not created by a naturaliy occurring biological process. A non-natural polymer Is a synthetic polymer that Is not made from naturaliy occurring (animal or plant) materials or monomers (such as amino acids, nucléotides, and saccharides). A polymer may be fully or partially natural, synthetic, or non-natural.

RNA1 Polynucleotide Conjugate We hâve found that conjugation of an RNAI poiynucleotide to a polynucleotide targeting moiety, either a hydrophobie group or to a galactose cluster, and co-administration of the RNAI polynucleotide conjugate with the delivery polymer described above provides for efficient, functional delivery of the RNAI polynucleotide to liver cells, particulariy hépatocytes, In vivo. By functional delivery, it Is meant that the RNAi polynucleotide Is delivered to the cell and has the expected biological activity, sequence-specific inhibition of gene expression. Many molécules, including polynucleotldes, administered to the vasculature of a mammal are normally cleared from the body by the liver. Clearance of a polynucleotide by the liver wherein the polynucleotide is degraded or otherwlse processed for removal from the body and wherein the polynucleotide does not cause sequence-specific inhibition of gene expression Is not considered functional delivery.

The RNAI polynucieotide conjugate is formed by covalently linking the RNAi polynucieotide to the polynucieotide targeting moiety. The polynucieotide Is synthesized or modified such that it contains a reactive group A. The targeting moiety Is also synthesized or modified such that it contains a reactive group B. Réactivé groups A and B are chosen such that they can be linked via a covalent linkage using methods known In the art.

The targeting moiety may be linked to the 3' or the 5' end of the RNAi polynucieotide. For siRNA polynucleotides, the targeting moiety may be linked to either the sense strand or the antisense strand, though the sense strand is preferred.

In one embodiment, the polynucieotide targeting moiety consists of a hydrophobie group More specifically, the polynucieotide targeting moiety consists of a hydrophobie group having at least 20 carbon atoms. Hydrophobie groups used as polynucieotide targeting moieties are herein referred to as hydrophobie targeting moieties. Exemplary suitable hydrophobie groups may be selected from the group comprislng: cholestérol, dicholesterol, tocopherol, ditocopherol, didecyl, didodecyl, dioctadecyl, didodecyl, dioctadecyl, isoprenoid, and choleamide. Hydrophobie groups having 6 or fewer carbon atoms are not effective as polynucieotide targeting moieties, while hydrophobie groups having 8 to 18 carbon atoms provide increasing polynucieotide delivery with Increasing size of the hydrophobie group (i.e. increasing number of carbon atoms). Attachment of a hydrophobie targeting moiety to an RNAI polynucieotide does not provide efficient functional in vivo delivery of the RNAI polynucieotide in the absence of coadministration of the delivery polymer. While siRNA-cholesterol conjugates hâve been reported by others to deliver siRNA (siRNA-cholesterol) to liver cells in vivo, in the absence of any additional delivery vehicle, high concentrations of siRNA are requlred and delivery efficacy is poor. When combined with the delivery polymers described herein, delivery of the polynucieotide is greatly improved. By providing the siRNA-cholesterol together with a delivery polymer of the Invention, efficacy of siRNA-cholesterol is increased about 100 fold.

Hydrophobie groups useful as polynucieotide targeting moieties may be selected from the group conslsting of: alkyl group, alkenyl group, alkynyi group, aryl group, aralkyl group, aralkenyl group, and aralkynyl group, each of which may be linear, branched, or cyclic, cholestérol, cholestérol dérivative, sterol, steroid, and steroïd dérivative. Hydrophobie targeting moieties are preferably hydrocarbons, containing only carbon and hydrogen atoms. However, substitutions or heteroatoms which maintain hydrophobicity, for example fluorine, may be permitted. The hydrophobie targeting moiety may be attached to the 3’ or 5' end of the RNAI polynucieotide using methods known In the art. For RNAI polynucleotides having 2 strands, such as siRNA, the hydrophobie group may be attached to either strand.

In another embodiment, the polynucleotide targeting molety comprises a galactose cluster (galactose cluster targeting molety). As used herein, a galactose cluster comprises a molécule having two to four terminal galactose dérivatives. As used herein, the term galactose dérivative Includes both galactose and dérivatives of galactose having affînity for the aslaloglycoprotein receptor equal to or greater than that of galactose. A terminal galactose dérivative Is attached to a molécule through Its C-1 carbon. The aslaloglycoprotein receptor (ASGPr) Is unique to hépatocytes and binds branched galactose-terminal glycoproteins. A preferred galactose cluster has three terminal galactosamines or galactosamine dérivatives each having affînity for the aslaloglycoprotein receptor. A more preferred galactose cluster has three terminal N-acetylgalactosamines. Other terms common In the art Include tri-antennary galactose, tri-valent galactose and galactose trimer. It Is known that tri-antennary galactose dérivative clusters are bound to the ASGPr with greater affînity than bi-antennary or mono-antennary galactose dérivative structures (Baenziger and Flete, 1980, Cell, 22, 611-620; Connoily et al., 1982, J. Biol. Chem., 257, 939-945). Mulivalency is required to achleve nM affînity. The attachment of a single galactose dérivative having affînity for the aslaloglycoprotein receptor does not enable functional delivery of the RNAI polynucleotide to hépatocytes In vivo when co-administered with the delivery polymer.

T OH

OH galactose

A galactose cluster contains three galactose dérivatives each linked to a central branch point. The galactose dérivatives are attached to the central branch point through the C-1 carbons of the saccharides. The galactose dérivative is preferably linked to the branch point via linkers or spacers. A preferred spacer Is a flexible hydrophilic spacer (U.S. Patent 5885968; Biessen et al. J. Med. Chem. 1995 Vol. 39 p. 1538-1546). A preferred flexible hydrophilic spacer Is a PEG spacer. A preferred PEG spacer Is a PEG₃ spacer. The branch point can be any small molécule which permits attachment of the three galactose dérivatives and further permits attachment of the branch point to the RNAi polynucleotide. An exemplary branch point group Is a di-lysine. A di-lysine molécule contains three amine groups through which three galactose dérivatives may be attached and a carboxyl reactive group through which the dl-iysine may be attached to the RNAI polynucleotide, Attachment of the branch point to the RNAI polynucleotide may occur through a linker or spacer. A preferred spacer Is a flexible hydrophllic spacer. A preferred flexible hydrophllic spacer Is a PEG spacer. A preferred PEG spacer is a PEG_} spacer (three ethyiene units). The galactose cluster may be attached to the 3' or 5’ end of the RNAi polynucleotide using methods known in the art. For RNAI polynucleotides having 2 strands, 5 such as siRNA, the galactose cluster may be attached to either strand.

A preferred galactose dérivative is an N-acetyl-galactosamine (GalNAc). Other saccharides having affinity for the asialoglycoproteln receptor may be selected from the list comprising: galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamlne, N-n-butanoylgalactosamine, and N-iso-butanoylgalactos-amine. The affinities of 10 numerous galactose dérivatives for the asialoglycoprotein receptor hâve been studied (see for example: iobst, S.T. and Drickamer, K. J.B.C. 1996, 271,6686) or are readily determined using methods typical in the art.

One embodiment of a Galactose cluster

HO_^N ο

Galactose cluster with PEG spacer between branch point and nucleic acid

The term polynucleotide, or nucleic acid or polynucleic acid, Is a term of art that refers to a polymer containing at least two nucléotides. Nucléotides are the monomeric units of polynucleotide polymers. Polynucleotides with less than 120 monomeric units are often called oligonucleotides. Naturel nucleic acids hâve a deoxyribose- or ribose-phosphate backbone. A non-naturel or synthetic polynucleotide is a polynucleotide that is polymerized in vitro or in a cell free System and contains the same or simiiar bases but may contain a backbone of a type other than the naturel ribose or deoxyribose-phosphate backbone. Polynucleotides can be synthesized using any known technique in the art. Polynucleotide backbones known in the art include: PNAs (peptide nucleic acids), phosphorothloates, phosphorodiamidates, morpholinos, and other variants of the phosphate backbone of native nucleic acids. Bases include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs. Synthetic dérivatives of purines and pyrimidines inciude, but are not lim'rted to, modifications which place new reactive groups on the nucléotide such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides. The term base encompasses any of the known base analogs of DNA and RNA. A polynucleotide may contain ribonucleotides, deoxyribonucieotides, synthetic nucléotides, or any suitable combination. Polynucleotides may be polymerized in vitro, they may be recombinant, contain chimeric sequences, or dérivatives of these groups. A polynucleotide may include a terminal cap moiety at the 5' -end, the 3’ -end, or both the 5’ and 3' ends. The cap moiety can be, but is not limited to, an inverted deoxy abasic moiety, an inverted deoxy thymidine moiety, a thymidine moiety, or 3' glyceryl modification.

An RNA interférence (RNAi) polynucleotide is a molécule capable of inducing RNA interférence through interaction with the RNA Interférence pathway machinery of mammalian cells to dégradé or Inhibit translation of messenger RNA (mRNA) transcripts of a transgene in a sequence spécifie manner, Two primary RNAI polynucleotides are small (or short) interfering RNAs (siRNAs) and micro RNAs (miRNAs). RNAI polynucleotides may be selected from the group comprising: siRNA, microRNA, double-strand RNA (dsRNA), short hairpin RNA (shRNA), and expression cassettes encoding RNA capable of inducing RNA interférence. siRNA comprises a double stranded structure typically contalnlng 15-50 base pairs and preferably 2125 base pairs and having a nucléotide sequence identical (perfectly complementary) or nearty Identical (partially complementary) to a coding sequence In an expressed target gene or RNA withln the cell. An siRNA may hâve dinucleotide 3' overhangs. An siRNA may be composed of two annealed polynucleotides or a single polynucleotide that forms a hairpin structure. An siRNA molécule of the Invention comprises a sense région and an antisense région. In one embodiment, the siRNA of the conjugate is assembled from two oligonucleotide fragments wherein one fragment comprises the nucléotide sequence of the antisense strand of the siRNA molécule and a second fragment comprises nucléotide sequence of the sense région of the siRNA molécule. In another embodiment, the sense strand is connected to the antisense strand vta a linker molécule, such as a polynucleotide linker or a non-nucleotide linker. MicroRNAs (miRNAs) are small noncoding RNA gene products about 22 nucléotides long that direct destruction or translational repression of their mRNA targets. If the complementarity between the miRNA and the target mRNA is partial, translation of the target mRNA is repressed. If complementarity is extensive, the target mRNA is cleaved. For miRNAs, the complex binds to target sites usually located In the 3’ UTR of mRNAs that typically share only partial homology with the miRNA. A “seed région - a stretch of about seven (7) consecutive nucléotides on the 5' end of the miRNA that forms perfect base pairing with its target - plays a key rôle In miRNA specifictty. Binding of the RISC/miRNA complex to the mRNA can lead to either the repression of protein translation or cieavage and dégradation of the mRNA. Recent data indicate that mRNA cieavage happens preferentialiy if there is perfect homology along the whole length of the miRNA and its target instead of showlng perfect base-palring only In the seed région (Pillai et al. 2007).

RNAI polynucleotide expression cassettes can be transcribed in the cell to produce small hairpin RNAs that can fonction as siRNA, separate sense and anti-sense strand linear siRNAs, or miRNA. RNA polymerase Üî transcribed DNAs contaln promoters selected from the list comprising: U6 promoters, H1 promoters, and tRNA promoters. RNA polymerase II promoters

Include U1, U2, U4, and U5 promoters, snRNA promoters, microRNA promoters, and mRNA promoters.

Llsts of known mlRNA sequences can be found In databases maintalned by research organizations such as Welicome Trust Sanger lnstitute, Penn Center for Bioinformatics, Memorial Sloan Kettering Cancer Center, and European Molecuie Biology Laboratory, among others. Known effective siRNA sequences and cognate bindlng sites are also well represented in the relevant iiterature. RNAI molécules are readily designed and produced by technologies known In the art. In addition, there are computational tools that Increase the chance of finding effective and spécifie sequence motifs (Pel et al. 2006, Reynolds et al. 2004, Khvorova et ai. 2003, Schwarz et al. 2003, ULTel et al. 2004, Heale et al. 2005, Chalk et al. 2004, Amarzguioui et al. 2004).

The polynucleotides of the invention can be chemlcally modified. Non-limlting examples of such chemical modifications Include: phosphorothloate internucleotide linkages, 2'-O-methyl ribonucleotldes, 2’-deoxy-2'-fluoro ribonucleotides, 2’-deoxy ribonucleotides, universal base nucieotides, 5-C-methyl nucléotides, and Inverted deoxyabasic residue incorporation. These chemical modifications, when used In various polynucléotide constructs, are shown to preserve polynucleotide actîvity In cells while at the same time Increasing the sérum stabllity of these compounds. Chemlcally modified siRNA can also minimlze the possibiiity of activating Interferon actîvity ln humans.

In one embodiment, a chemically-modified RNAI polynucleotide of the Invention comprises a duplex having two strands, one or both of which can be chemically-modified, wherein each strand Is about 19 to about 29 nucieotides. In one embodiment, an RNAI polynucleotide of the invention comprises one or more modified nucieotides while maintalnlng the ability to médiate RNAI Inside a cell or reconstituted in vitro System. An RNAI polynucleotide can be modified wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the nucieotides. An RNAI polynucleotide of the Invention can comprise modified nucieotides as a percentage of the total number of nucieotides présent in the RNAI polynucleotide. As such, an RNAI polynucleotide of the Invention can generally comprise modified nucieotides from about 5 to about 100% of the nucléotide positions (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%. 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%, 90%, 95% or 100% of the nucléotide positions). The actual percentage of modified nucieotides présent in a given RNAI polynucleotide dépends on the total number of nucieotides présent In the RNAI polynucleotide. If the RNAi polynucleotide is single stranded, the percent modification can be based upon the total number of nucieotides présent In the single stranded RNAI polynucleotide. Ukewise, if the RNAI polynucleotide Is double stranded, the percent modification can be based upon the total number of nucléotides présent In the sense strand, antisense strand, or both the sense and antisense strands. In addition, the actual percentage of modified nucléotides présent in a given RNAI polynucleotide can also dépend on the total 5 number of purine and pyrimidïne nucléotides présent in the RNAi polynucleotide. For example, wherein ali pyrimidine nucléotides and/or ail purine nucléotides présent in the RNAI polynucleotide are modified.

An RNAI polynucleotide modulâtes expression of RNA encoded by a gene. Because multiple genes can share some degree of sequence homology with each other, an RNAI polynucleotide 10 can be designed to target a class of genes with suffirent sequence homology. Thus, an RNAi polynucleotide can contaln a sequence that has complementarity to sequences that are shared amongst different gene targets or are unique for a spécifie gene target. Therefore, the RNAi polynucleotide can be designed to target conserved régions of an RNA sequence having homology between several genes thereby targeting several genes In a gene family (e.g., 15 different gene isoforms, splice variants, mutant genes, etc.). In another embodiment, the RNAi polynucleotide can be designed to target a sequence that is unique to a spécifie RNA sequence of a single gene.

The term complementarity refers to the ability of a polynucleotide to form hydrogen bond(s) with another polynucleotide sequence by either traditional Watson-Crick or other non-traditional 20 types. In reference to the polynucleotide molécules of the présent invention, the binding free energy for a polynucleotide molécule with Its target (effector binding site) or complementary sequence is sufficient to allow the relevant fonction of the polynucleotide to proceed, e.g., enzymatic mRNA cleavage or translation inhibition. Détermination of binding free energies for nucleic acid molécules is well known in the art (Frier et al. 1986, Turner et al. 1987). A percent 25 complementarity Indicates the percentage of bases, In a contiguous strand, in a first polynucleotide molécule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second polynucleotide sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). Perfectiy complementary means that ail the bases in a contiguous strand of a polynucleotide sequence will hydrogen bond with the same number of 30 contiguous bases in a second polynucleotide sequence.

By inhlbit, down-regulate, or knockdown gene expression, it is meant that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein or protein subunit translated from the RNA, Is reduced below that observed in the absence of the blocking polynucleotide-conjugates of the Invention. Inhibition, down-regulation, 32 or knockdown of gene expression, with a polynucleotide delivered by the compositions of the invention, is preferably below that level observed In the presence of a control inactive nucleic acid, a nucleic acid with scrambled sequence or with inactivating mlsmatches, or in absence of conjugation of the polynucleotide to the masked polymer.

In Vivo Administration

In pharmacology and toxicology, a route of administration Is the path by which a dru g, fluid, poison, or other substance 1s brought into contact with the body. In general, methods of administering drugs and nucleic acids for treatment of a mammal are well known In the art and can be applied to administration of the compositions of the invention. The compounds of the présent Invention can be administered via any suitable route, most preferably parenterally, In a préparation appropriately tailored to that route. Thus, the compounds of the présent invention can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, or intraperitoneally. Accordingly, the présent Invention also provides pharmaceutical compositions comprislng a pharmaceutically acceptable carrier or excipient.

Parentéral routes of administration include intravascular (intravenous, intraarterial), intramuscular, intraparenchymal, Intradermai,’ subdermai, subcutaneous, intratumor, intraperitoneal, Intrathecal, subdural, épidural, and intralymphatic injections that use a syringe and a needle or cathéter. Intravascular herein means within a tubular structure called a vessel that Is connected to a tissue or organ within the body. Within the cavity of the tubular structure, a bodily fluid flows to or from the body part. Examples of bodily fluid include blood, cerebrospinal fluid (CSF), lymphatic fluid, or bile. Examples of vessels Include arteries, artérioles, capillaries, venules, sinusoids, veins, lymphatics, bile ducts, and ducts of the salivary or other exocrine glands. The intravascular route indudes delivery through the blood vessels such as an artery or a vein. The blood circulatory System provides systemlc spread of the pharmaceutical.

The described compositions are injected in pharmaceutically acceptable carrier solutions. Pharmaceutically acceptable refers to those properties and/or substances which are acceptable to the mammal from a pharmacological/toxicological point of view. The phrase pharmaceutically acceptable refers to molecular entities, compositions, and properties that are physiologically tolerable and do not typically produce an allergie or other untoward or toxic reaction when administered to a mammal. Preferably, as used herein, the term pharmaceutically acceptable means approved by a regulatory agency of the Fédéral or a state govemment or iisted in the U.S. Pharmacopela or other generally recognized pharmacopeia for use in animais and more particularly in humans.

The RNAi poiynucleotide-targeting moiety conjugale Is co-administered with the delivery poiymer. By co-admlnistered it Is meant that the RNAi polynucleotide and the delivery poiymer are admlnlstered to the mammal such that both are présent in the mammal at the same time. The RNAi poiynucleotide-targeting moiety conjugale and the delivery poiymer may be 5 administered simultaneously or they may be delivered sequentially. For simultaneous administration, they may be mixed prior to administration. For sequential administration, either the RNAI poiynucleotide-targeting moiety conjugale or the deiivery poiymer may be administered first

For RNAi polynucleotide-hydrophoblc targeting moiety conjugates, the RNAI conjugate may be 10 administered up to 30 minutes prior to administration of the delivery poiymer. Also for RNAI polynucleotide-hydrophobic targeting moiety conjugates, the deiivery poiymer may be administered up to two hours prior to administration of the RNAi conjugate.

For RNAi poiynucleotide-galactose cluster targeting moiety conjugates, the RNAI conjugate may be administered up to 15 minutes prior to administration of the delivery poiymer. Also for 15 RNAi poiynucleotide-galactose cluster targeting moiety conjugates, the delivery poiymer may be administered up to 15 minutes prior to administration of the RNAi conjugate.

Therapeutic Effect

RNAi polynucleotides may be delivered for research purposes or to produce a change In a celi that is therapeutic. In vivo deiivery of RNAi polynucleotides is useful for research reagents and 20 for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications. We hâve disclosed RNAI polynucleotide deiivery resulting in inhibition of endogenous gene expression in hépatocytes. Levels of a reporter (marker) gene expression measured following delivery of a polynucleotide Indicate a reasonable expectation of slmiiar levels of gene expression following delivery of other polynucleotides. Levels of treatment 25 considered bénéficiai by a person having ordinary skill in the art differ from disease to disease.

For example, Hemophiiia A and B are caused by deficiencies of the X-iinked clotting factors Vlii and IX, respectively. Their clinical course is greatiy influenced by the percentage of normal sérum levels of factor Viil or IX: < 2%, severe; 2-5%, moderate; and 5-30% mild. Thus, an increase from 1% to 2% of the normal level of circulating factor In severe patients can be 30 considered bénéficiai. Levels greater than 6% prevent spontaneous bleeds but not those secondary to surgery or injury. Slmilariy, inhibition of a gene need not be 100% to provide a therapeutic benefit. A person having ordinary skili in the art of gene therapy would reasonably anticipate bénéficiai ieveis of expression of a gene spécifie for a disease based upon sufficient levels of marker gene results. In the hemophiiia example, if marker genes were expressed to yield a protein at a level comparable in volume to 2% of the normal level of factor VIII, It can be reasonably expected that the gene coding for factor VIII would also be expressed at slmllar levels. Thus, reporter or marker genes serve as useful paradigme for expression of intracellular proteins in general.

The liver is one of the most important target tissues for gene therapy given its central rôle in metabolism (e.g., lipoproteln metabolism in various hypercholesteroiemias) and the sécrétion of circulating proteins (e.g., clotting factors in hemophilia). In addition, acquired disorders such as chronic hepatitis and cirrhosis are common and are also potentially treated by polynucleotidebased liver thérapies. A number of diseases or conditions which affect or are affected by the liver are potentially treated through knockdown (inhibition) of gene expression in the liver. Such liver diseases and conditions may be selected from the list comprising: liver cancers (including hepatocellular carcinoma, HCC), viral infections (including hepatitis), metabolic disorders, (including hyperlipidemia and diabètes), fibrosis, and acute liver injury.

The amount (dose) of delivery polymer and RNAi-polynucleotide-conjugate that ls to be adminlstered can be determined empirically. We hâve shown effective knockdown of gene expression using 0.1-10 mg/kg animal weight of siRNA-conjugate and 5-60 mg/kg animal weight delivery polymer. A preferred amount in mice is 0.25-2.5 mg/kg siRNA-conjugate and 10-40 mg/kg delivery polymer. More preferably, about 12.5-20 mg/kg delivery polymer is adminlstered. The amount of RNAi polynucleotide-conjugate is easily increased because it is typically not toxic in larger doses.

As used herein, in vivo means that which takes place inside an organism and more specifically to a process performed in or on the living tissue of a whole, living multicellular organism (animal), such as a mammal, as opposed to a partial or dead one.

Examples

Polymer Synthèses

Example 1. Po!y(vinyl ether) random copolymers.

Vinyl ether monomers for incorporation of amine-containlng monomers. 2Vinyloxy Ethyî Phthalimide was prepared via reactlng 2-chloroethyl vinyl ether (25 g, 0.24 mol; CAS #110-75-8) and potassium phthalimide (25 g, 0.135 mol; CAS #1074-82-4) in 100°C N,N-Dimethylformamlde (DMF, 75 ml) using tetra n-butyl ammonium bromide (0.5 g; CAS #1643-19-2) as the phase transfer catalyst. This solution was heated for 6 h and then crashed out in water and filtered. This solîd was then recrystallized twice from methanol to give white crystals.

Synthesis of water-sotuble, amphipathic, membrane active polyfvinyl ether) polyamine terpolymers. X mol% amine-protected vinylether (e.g., 2-Vinyloxy Ethyl Phthalimide) Is added to an oven dried round bottom flask under a blanket of nitrogen in anhydrous dichloromethane. To this solution Y mol% lower hydrophobie group (e.g., propyl, butyl) vinylether and optionally Z mol% higher hydrophobie group (e.g., dodecyl, octadecyl) vinylether are added (FIG. 1). The solution is placed In a -50 to -78°C bath, and the 2-vinyloxy ethyi phthalimide Is allowed to preclpitate. To this solution 10 mol % BF₃-(OCH₂CH₃)₂ is added and the reaction is allowed to proceed for 2-3 h at -50 to -78°C. Polymerization is terminated by addition of ammonium hydroxide In methanol solution. The polymer Is brought to dryness under reduced pressure and then brought up in 1,4-dioxane/methanol (2/1). 20 mol eq. of hydrazine per phthalimide is added to remove the protecting group from the amine. The solution is refluxed for 3 h and then brought to dryness under reduced pressure. The resulting soiid Is dissolved In 0.5 mol/L HCl and refluxed for 15-min to form the hydrochloride sait of the polymer, diluted with distilled water, and refluxed for an additional hour. The solution is then neutralized with NaOH, cooled to room température (RT), transferred to molecular cellulose tubing, dialyzed agalnst distilled water, and lyophilized. The polymer can be further purified using size exclusion or other chromatography. The molecular weight of the polymers Is estimated using columns according to standard procedures, Including analytical size-exclusion chromatography and size-exclusion chromatography with multi-angle light scattering (SEC-MALS).

Synthesis of DW1360. An amine/butyl/octadecyl poly(vinyi ether) terpolymer, was synthesized from 2-vinyloxy ethyl phthalimide (5 g, 23.02 mmol), butyl vinylether (0.665 g, 6.58 mmol), and octadecyl vinylether (0.488 g, 1.64 mmol) monomers. 2-vinyloxy ethyl phthalimide was added to a 200 mL oven dried round bottom flask containing a magnetic stir bar under a blanket of Argon In 36 mL anhydrous dichloromethane. To this solution was added butyl vinyi ether and n-octadecyt vinyi ether. The monomers were fully dissolved at room température (RT) to obtain a clear, homogenous solution. The reaction vessel containing the clear solution was then placed into a -50’C bath generated by addition of dry ice to a 1:1 solution of ACS grade denatured atcohot and ethylene glycol and a visible précipitation of phthalimlde monomer was allowed to form. After coolîng for about 1.5 min, BF3 (OCH₂CHj)2 (0.058 g, 0.411 mmol) 5 was added to Initiale the potymerization reaction. The phthalimlde monomer dissolved upon initiation of polymerization. The reaction was allowed to proceed for 3 h at -50*C. The polymerization was stopped by the addition of 5 mL of 1% ammonium hydroxide in methanol. The solvents were then removed by rotary évaporation.

The polymer was then dissolved in 30 mL of 1,4-dîoxane/methanol (2/1). To this solution was added hydrazine (0.147 g, 46 mmol) and the mixture was heated to reflux for 3 h. The solvents were then removed by rotary évaporation and the resulting solid was then brought up in 20 mL of 0.5 mot/L HCl and refluxed for 15 minutes, diluted with 20 mL distilled water, and refluxed for t5 an additional hour. This solution was then neutralized with NaOH, cooled to RT, transferred to

3,500 molecular weight cellulose tubing, diatyzed for 24 h (2*20L) against distilled water, and lyophitized.

While polymers containing the indicated vinyl ether monomers are described, the invention is 20 not limited to these particular monomers.

D. Synthesis of water-soluble, amphlpathlc, membrane active poly(acrylate) polyamine terpolymers Poty(acrylate) and poly(methytacrylate) heteropolymers may be synthesized using the general free radical reaction scheme (as used herein a poly(methacrylate) polyamine Is a 25 subgenus of the genus poly(acrytate) polyamine):

catalyst

wherein R is independently a hydrogen or methyl group and X represents the desired monomer pendent groups présent In the polymer at the desired ratios.

For polymer synthèses, suitable monomers include, but are not limited to: BOC-protected amine-containing monomers (M):

O N—BOC

Ο wherein η = 1-4 and removal of the BOC protecting group yields a primary amine.

Lower hydrophobie group monomers (N):

R ⁰ wherein n = 1-5 and one or more carbons may be unsaturated.

Higher hydrophobie group monomers (0):

R

O wherein n = 8-24 and one or more carbons may be unsaturated.

Using the above monomers, membrane active heteropolymers can be syntheslzed with the following compositions: M can be 50-90 mol%; N can be 10-50 mol %; O can be 0-10 mol%.

E. Synthesls ofwater-soluble, amphipathic, membrane active poly(acrylate) polyamine terpolymers.

TFA (BOC removal)

R, R', and R are independently hydrogen or methyl x = 2,3, or 4 y = 0,1, 2, 3,4, or 5 [methyi (C1) - hexyi (C6)J z = integer^ 8 [decyl (C10) or greater] a, b, and d are integers selected such that the polymer has the deslred ratio of monomers as described above.

X mol% amine-protected acrylate monomer, Y mol% iower hydrophobie group acrylate monomer, and optionally 2 mol% higher hydrophobie group acrylate monomer are added to a reaction tube equipped with a stir bar. An appropriate solvent (e.g., acetonitriie or dioxane) Is added, followed by an appropriate catalyst (e,g„ AIBN), and the réaction mixture Is purged with N₂. The reaction tubes are then capped and transferred to an oîl bath and heated (e.g., 60°C) for sufficient time to allow polymerization (e.g., 3 h). The crude polymer may be purified by appropriate means, inciuding but not limited to dialysis, column chromatography, and précipitation, prior to removal of the BOC protecting groups. The BOC protecting groups are removed by reaction with 2M HCl In glacial acetic acid. Removal of the BOC protecting groups yleld polymer primary amines and a water soluble membrane active poly(acrylate) polyamine. The polymer may then be purified by appropriate means, inciuding dialysis, column chromatography, and précipitation.

Synthesis of (Ant 40911-3 23-28, Ant 40911-35-2). 2,2’-Azobis(2-methylpropionitrÎle) (AIBN, radical Initiator), acetonitrile, and dioxane were purchased from Sigma Aldrich. Acrylate and méthacrylate monomers were filtered to remove inhibitors. 3-(BOC-amino)1-propanol (TCI) was reacted with acryloyl chloride (CAS 614-66-6) to produce BOC-amino propyl acrylate (ΒΑΡΑ).

ΒΑΡΑ in a 2L round-bottom flask equîpped with a stir bar, 2-(2-aminoethoxy) éthanol (21.1g, 202.9 mmol) was dissolved In 350 mL dichloromethane. In a separate 1L flask, BOC anhydride (36.6g, 169.1 mmol) was dissolved In 660 mL dichloromethane. The 2L round-bottom flask was fitted with an addition funnel and BOC anhydride solution was added to the flask over 6 h. The reaction was left to stir overnîght. In a 2L separatory funnel, the product was washed with 300 ml each of 10% citric acid, 10% K₂CO₃, sat. NaHCO₃, and sat. NaCl. The product, BOC protected 2-(2-aminoethoxy) éthanol, was dried over Na₂SO₄, gravity filtered, and DCM was evaporated using rotary évaporation and high vacuum.

In a 500 ml round bottom flask equipped with a stir bar and flushed with argon, BOC protected 2-(2-aminoethoxy) éthanol (27.636g, 135.8 mmol) was added, followed by 240 mL anhydrous dichloromethane, Diisopropylethyl amine (35.5 ml, 203.7 mmol) was added, and the System was placed In a dry Ice/acetone bath. Acryloyl Chloride (12.1 ml, 149.4 mmol) was diluted using 10 ml of dichloromethane, and added drop-wise to the argon flushed System. The System was kept under argon and left to corne to room température and stirred overnight. The product was washed with 100 mL each of dH₂O, 10% citric acid, 10% K₂CO₃, sat. NaHCO₃, and saturated NaCl. The product, BOC-amino ethyl ethoxy acrylate (BAEEA), was dried over Na₂SO₄, gravity filtered, and DCM was evaporated using rotary évaporation. The product was purified through column chromatography on 29 cm silica using a 7.5 cm diameter column. The solvent System used was 30% ethyl acetate in hexane. Rf: 0.30. Fractions were collected and solvent was removed using rotary évaporation and high vacuum. BAEEA, was obtained with 74% yield. BAEEA was stored In the freezer.

BAEEA

Polymer 40911-3 23-28: 70% ΒΑΡΑ, 25% butyl méthacrylate (CAS 97-88-1), 5% octadecyl méthacrylate (CAS 4813-57-4), (3% AIBN catalyst) mole feed ratio (0.0139 total mol). ΒΑΡΑ 5 (9.739 mmol) (A), butyl méthacrylate (3.478 mmol) (B), and octadecyl méthacrylate (0.6957 mmol) (D) were added to a 20 mL reaction tube equipped with a stir bar. Acetonitrile (16 ml) was added, followed by AIBN (0.4174 mmol). The above steps were repeated In order to hâve two reactions run In tandem. The reaction mixture was purged with N₂ for 30 min. The reaction tubes were then capped and transferred to an oil bath and heated at 60'C for 3 h. The tubes 10 were removed and the contents were combined. The crude polymer was precipitated into delonized water, and reacted with neat trifluoroacetic acid (40 ml) for 1.5 h to remove the BOC protecting groups and produce the primary amines and a water soluble membrane active poly(acrylate) polyamlne. 200 mL deionized H₂O (dH₂O) were added to the reaction, the solution was transferred to 3500 MW cutoff cellulose tubing, dialyzed against high sait for 24 h, 15 then against dH₂O for 18 h. The contents were evaporated to dryness, dissolved in 100 mL dH₂O and lyophilized. The dried polymer was dissolved in 50% MeOH/100 mM ammonium formate/0.2% formic acid solution at 25 mg/ml. Three injections of crude polymer solution (250 mg, 10 ml) were purified on S-200 sephacryl media using an XK50/30 cm column used at a flow rate of 5.0 ml/min. The column was packed and used according to the manufacturées 20 instructions. (GE Healthcare, instructions 56-1130-82 Al, 52-2086-00 AK). Polymer elution was detected using a Shimadzu RID-10A refractive index collecter. Fractions from 23 min to 28 min were collected and combined for each run. The solvent was evaporated and the purified polymer was lyophilized twice.

Polymer Ant 40911-35-2: 80% BAEEA, 15% butyl methacryiate, 5% octadecyl acrylate, (3% AIBN catalyst) mole feed ratio (0.013913 total mol). BAEEA (A) (11.13 mmol), butyl methacryiate (B) (2.086 mmol), and octadecyl acrylate (D) (0.6957 mmol) were added to a 20 mL reaction tube equipped with a stir bar. Dioxane (16 ml) was added, followed by AIBN (0.4174 mmol). The above steps were repeated In order to hâve two reactions run in tandem.

The reaction mixture was purged with N₂ for 30 min. The reaction tubes were then capped and transferred to an oil bath and heated at 60“C for 3 h. The tubes were removed and the contents were combined. Dioxane was evaporated through rotary évaporation and hlgh vacuum and the crude polymer was dissolved in 89.8% dichloromethane/10% tetrahydrofuran/0.2% triethylamine solution at 70 mg/ml. Three injections of crude polymer solution (700 mg, 10 ml) 35 were purified on a Jordi gel divinyl benzene 10« Λ column (internai diameter: 22 mm, length:

500 mm) used at a flow rate of 5.0 ml/min. Polymer elution was detected using a Shimadzu

RID-10A refractive index collector. Fractions from 15.07 mln-17.13 min were collected and combined. The solvent was evaporated through rotary évaporation.

Approximately 10 mg of the polymer was dissolved in 0.5 mL 89.8% dîchloromethane, 10% tetrahydrofuran, 0.2% triethylamine. The molecular weight and polydispersity (PDI) were measured uslng a Wyatt Helos II multiangle light scattering detector attached to a Shimadzu Prominence HPLC using a Jordi 5μ 7.8*300 Mixed Bed LS DVB column. A molecular weight of 172,000 and a PDI of 1.26 were obtained.

The purified BOC-protected polymer was reacted with neat trifluoroacetic acid (7 ml) for 1.5 h (or 2 M HCl in glacial acetic acid for 0.5 h) to remove the BOC protecting groups and produce the amines. 40 mL dH₂O were added to the reaction, the solution was transferred to 3500 MW cutoff cellulose tubing, dialyzed against high sait for 24 hr, then against dH₂O for 18 h. The contents were evaporated to dryness, then dissolved in 20-30 mL dH₂O and lyophilized twice. The polymer solution was stored at 2-8’C.

The number of carbon atoms linking the amine to the backbone of the polymer and whether or not the linker is branched, affects the pKa of the amine and steric effects near the amine. For example, for the above polymers, ethyî amine has a pKa of about 8.1, propyl amine has a pKa of about 9.3, and pentyl amine has a pKa of about 10.2. The pKa of the amine or steric effects near the amine affect the lability of masking groups attached to the amine. For réversible attachaient of a maleic anhydride to an amine, a higher pKa of the amine results Is a slower rate of release of an anhydride from the amine. Also, increased steric hindrance near the amine, such as with an isopropyl linker, may increase the pKa of the amine.

Polymer Lau 41305-38-17-19: 80% ΒΑΡΑ, 20% ethyî méthacrylate (CAS 97-63-2), (3% AIBN catalyst) mole feed ratio (0.0105 total mol). ΒΑΡΑ (A) (8.40 mmol) and ethyî méthacrylate (B) (2.10 mmol) were added to a 15 mL reaction tube equipped with a stîr bar. Acetonitrile (11.5 ml) was added followed by AIBN (0.315 mmol). The above steps were repeated in order to hâve two reactions run in tandem. The reaction mixture was purged with N₂ for 30 min. The reaction tubes were then capped and transferred to an oil bath and heated at 60’C for 3 h. The tubes were removed and the contents were combined. Acetonitrile was evaporated through rotary évaporation and high vacuum and the crude polymer was dissolved in 74,8% dîchloromethane / 25% tetrahydrofuran/0.2% triethylamine solution at 50 mg/ml, Three injections of crude polymer solution (500 mg, 10 ml) were purified on a Jordi gel fluorinated divlnyl benzene 10* A column (internai diameten 22 mm, length: 500 mm) used at a flow rate of 5.0 ml/min. Polymer elution 42 was detected using a Shimadzu RID-10A refractive index collector. Fractions from 17.16 min19.16 min were collected and comblned. The solvent was evaporated through rotary évaporation. The purified BOC-protected polymer was reacted with 2M HCl In glacial acetic acid (7 ml) for 1.5 h to remove the BOC protecting groups and produce the amines. 40 mL dH₂O 5 were added to the reaction, the solution was transferred to 3500 MW cutoff cellulose tubing, diaiyzed against high sait for 24 hr, then agalnst dH₂O for 16 h. The contents were evaporated to dryness, then dissoived in 30 mL dH₂O and lyophilized twice.

F. Similar polymers, synthesized from (protected) amine monomers, lower hydrophobie group !0 monomers, and higher hydrophobie group octadecyl groups would be predicted to be effective in the practice of the described invention.

Polymer Characterization

Ex ample 2. Characterization ofDW1360.

!5 Amphipathic analysis. 1,6-diphenyl-1,3,5-hexatriene (DPH, Invitrogen) fluorescence (λ._χ = 350 nm; A,_m = 452 nm) is enhanced in a hydrophobie environment. This fluorophore was used to analyze the DW1360 polymer. 0.5 μΜ (final concentration) DPH was added to 10 pg DW1360 In 0.5 mL 50 mM HEPES buffer, pH 8.0. The solution was then tested for DPH accumulation in a 20 hydrophobie environment by measuring fluorescence of DPH. Increased DPH fluorescence in the presence of the conjugates indicates the formation of a hydrophobie environment by the polymer.

Molecular Weight. Polymer Molecular Weights (mass) (MW) were determined on a Wyatt Dawn Heleos II run in conjunction with optilab rEX in batch mode. Polymers was brought up at varying concentrations in appropriate solvent and each was loaded onto the Wyatt system. Astra software then calculated changes in refractive Index as a fonction of concentration (dn/dc) which was used in a Zimm piot to calculate MW. The average molecular weight determined for purified DW1360 was 4000-6000 Da. The average molecular weight for the purified acrylate polymers was about 100-120 kDa.

Particle Slzing and Zêta Potentiel. The zêta potential of the polymers was measured using a Malvem Zetasizer nano sériés (Nano ZS) instrument. The 35 zêta potential of the CDM-masked polymers varied between 0 and -30 mV and more predominantly between 0 and -20 mV. Zêta potential was measured In isotonie glucose buffered at pH 8 with residual HEPES. At pH 7, the conjugales would be expected to gain some positive charge due to protonation of some of the amines.

Quantification of amine groupa in conjugate after CDM-reagent modification. DW1360 polymer was synthesized as described previously followed by treatment with 14 wt équivalents HEPES base and 7 wt équivalents of a 2:1 wt:wt mixture of CDM-NAG and CDM-PEG (average 11 units). One hour iater, the amine content of the maleic anhydride dérivative treated conjugate was measured by treatment with trinitrobenzene sulfonic acid (TNBS) in 100 mM NaHCOl· When normalized to a conjugate that had not been maleamate modified, It was determined that the amount of modified amines was about 75% of total. This degree of modification may be varied by changing the amount of added maleic anhydride or altering the reaction conditions.

Uposome lysls. 10 mg of egg phosphatidylcholine was hydrated with 1 mL of buffer containing 100 mM carboxyfluorescein (CF) and 10 mM HEPES pH 7.5. Liposomes were then be extruded through 100-nm pores polycarbonate filters (Nucleopore, Pleasanton, CA). Unentrapped CF was removed by size exclusion chromatography using Sepharose 4B-200 eluting with 10 mM HEPES at pH 8 and 0.1 mol/L NaCI. A 200 pL aliquot of the CF-loaded liposomes were added to 1.8 mL of isotonie buffer. Fluorescence (O_ex=488, □_βπι=540) was measured 30 min after addition of 0.25 pg of polymers to vesicle suspensions. At the end of each experiment, vesicles were disrupted by the addition of 40 pl of a 1% Triton X-100 solution to détermine maximal lysis.

Polymer Masking Agents

Example 3. Masking agents.

Synthesis of 2-propionic-3-methyfmaielc anhydride masking agent precursor (carboxydimethylmaleic anhydride orCDM).

OH

2-propionic-3-methylmaleic anhydride

To a suspension of sodium hydride (0.58 g, 25 mmol) in 50 mL anhydrous tetrahydrofuran was added triethyl-2-phosphonopropionate (7.1 g, 30 mmol). After évolution of hydrogen gas had stopped, dimethyl-2-oxoglutarate (3.5 g, 20 mmol) in 10 mL anhydrous tetrahydrofuran was added and stirred for 30 min. 10 mL water was then added, and the tetrahydrofuran was 5 removed by rotary évaporation. The resuiting solid and water mixture was extracted with 3x50 mL ethyl ether. The ether extractions were combined, dried with magnésium sulfate, and concentrated to a light yellow oil. The oil was purified by silica gel chromatography elution with 2:1 etherihexane to yield 4 g (82% yleld) of pure triester. The 2-propionic-3-methylmaieic anhydride was then formed by dissolving of this triester into 50 mL of a 50/50 mixture of water 10 and ethanoi containing 4.5 g (5 équivalents) of potassium hydroxide. This solution was heated to reflux for 1 h. The éthanol was then removed by rotary évaporation and the solution was acldified to pH 2 with hydrochloric acid. This aqueous solution was then extracted with 200 mL ethyl acetate, isolated, dried with magnésium sulfate, and concentrated to a white solid. This solid was then recrystallized from dichloromethane and hexane to yield 2 g (80% yield) of 15 2-propionic-3-methylmaleic anhydride.

Thioesters, esters, and amides may be synthesized from CDM by conversion of CDM to its acid chloride with oxalyl chloride followed by the addition of a thlol, ester, or amine and pyridine. CDM and its dérivatives are readily modified, by methods standard in the art, with targeting 20 ligands, steric stabilizers, charged groups, and other reactive groups. The résultant molécules can be used to reverslbly modify amines.

Masking agents were synthesized through modification of CDM to produce preferably charge neutral agents:

wherein R1 comprises an ASGPr targeting ligand or steric stabilizer (e.g. PEG).

B. Masking Agent containing an ASGPr targeting group. The most widely-studied hépatocyte targeting ligands are based on galactose, which is bound by the asialoglycoprotein receptor 30 (ASGPr) on hépatocytes. Attachment of galactose or a galactose dérivative has been shown to facilitate hépatocyte targeting of a few highly water soluble, uncharged polymers, including: the oligosaccharide chitosan, a polystyrène dérivative, and a polyacrylamide HPMA. ASGPr targeting groups are readily generated using lactose, a galactose-glucose disaccharide, via modification of the glucose residue. Lactobionic acid (LBA, a lactose dérivative in which the glucose has been oxidized to gluconic acid) is readily incorporated Into a maleic anhydride dérivative using standard amide coupling techniques.

C. Steric stabilizer CDM-PEG and targeting group CDM-NAG (N-acetyl galactosamine) synthèses. To a solution of CDM (300 mg, 0.16 mmol) In 50 mL methylene chloride was added oxalyi chloride (2 g, 10 wt, eq.) and dimethylformamide (5 μΙ). The reaction was allowed to proceed overnight, after which the excess oxalyi chloride and methylene chloride were removed by rotary évaporation to yield the CDM acid chloride. The acid chloride was dissoived in 1 mL of methylene chloride. To this solution was added 1.1 molar équivalents polyethylene glycol monomethyl ether (MW average 550) for CDM-PEG or (aminoethoxy)ethoxy-2-(acetylamlno)-2deoxy-p-D-galactopyranoside (i.e. amîno bisethoxyl-ethyl NAG) for CDM-NAG, and pyridine (200 μΙ, 1.5 eq) In 10 mL of methylene chloride. The solution was then stirred 1.5 h. The solvent was then removed and the resulting solid was dissoived Into 5 mL of water and purified using reverse-phase HPLC using a 0.1% TFA water/acetonitrile gradient.

CDM-PEG

Preferably, PEG containing from 5 to 20 ethylene units are attached to the di-substituted maleic 25 anhydride. More preferably, PEG containing 10-14 ethylene units are attached to the disubstituted maleic anhydride. The PEG may be of variable length and hâve a mean length of 5 or 10-14 ethylene units. Alternative^, the PEG may be monodisperse, uniform or discrète; having, for example, exactly 11 or 13 ethylene units.

CDM-NAG

As shown above, a PEG spacer may be positioned between the anhydride group and the ASGPr targeting group, A preferred PEG spacer contains 1-10 ethylene units.

CDM-NAG with alkyl spacer

Réversible Polymer Modification

Example 4. Réversible modification/masking of membrane active polyamine; l.e., modification of membrane active polymer with CDM-NAG or a mixture of CDM-NAG plus CDM-PEG. To a solution of * mg membrane active polyamine (e.g. DW1360 described above) in isotonie glucose was added 14* mg of HEPES free base followed by either 7x mg CDM-NAG or a mixture of 2.3* mg CDM-NAG and 4.6* mg CDM-PEG, for a total of 7* disubstituted maleic anhydride masking agent. The solution was then incubated for at least 30 min at RT prior to animal administration. Reaction of CDM-NAG or CDM-PEG with the polyamine yielded:

wherein R Is the polymer and R1 comprises a ASGPr targeting moiety or steric stabilizer. The anhydride carboxyl produced in the reaction between the anhydride and the polymer amine exhibits -1/20⁹¹ of the expected charge (Rozema et al. Bioconjugate Chemistry 2003). Therefore, the membrane active polymer Is effectively neutralized rather than being converted to a highly negatively charged polyanion.

siRNA-conjugate

Example 5. RNAI polynucleotide-targeting moiety conjugales.

siRNA-hydrophobe conjugale. Various hydrophobie groups were covalently linked to 3* or 5' ends of siRNA molécules using techniques standard in the art.

siRNA-GalNAc cluster conjugale. The GalNAc cluster was made by attachaient of three GalNAc PEGa groups to the amines on a di-lysine branch point. The carboxyl group on the di-lysine is then available for covalent attachaient to the RNAi polynucleotide, such as an siRNA.

In Vivo siRNA Delivery

Example 6. Administration of RNAI polynucleotides In vivo, and delivery to hépatocytes. RNAI 20 polynucleotide conjugates and masked polymers were synthesized as described above. Six to eight week old mice (strain C57BL/6 or ICR, -18-20 g each) were obtained from Harlan

Sprague Dawiey (Indianapolis IN). Mice were housed at least 2 days prior to Injection. Feeding was performed ad libitum with Hartan Teklad Rodent Diet (Harlan, Madison Wl). RNAi polynucleotide conjugales and masked polymers were synthesized as described above. Mice were injected with 0.2 mL solution of delivery polymer and 0.2 mL siRNA conjugates Into the tail vein. For slmultaneous injection of polymer and siRNA, the siRNA-conjugate was added to modified polymer prior to injection and the entîre amount, 0.4 ml, was Injected. The composition was soluble and nonaggregating in physiological conditions. For Injections in which polymer and siRNA are injected separately, polymer was injected in 0.2 mL of formulation solution and siRNA was injected in 0.2 mL of isotonie glucose. Solutions were Injected by infusion into the tail vein. Injection into other vessels, e.g. retro-orbital Injection, were equally effective.

Sérum ApoB levels détermination. Mice were fasted for 4 h (16 h for rats) before sérum collection by submandibular bleeding. Sérum ApoB proteln levels were determined by standard sandwich ELISA methods. Briefly, a polyclonal goat anti-mouse ApoB antibody and a rabbit anti-mouse ApoB antibody (Biodesign International) were used as capture and détection antibodies respectively. An HRP-conjugated goat anti-rabbit IgG antibody (Sigma) was applied afterwards to blnd the ApoB/antibody complex. Absorbance of tetramethyl-benzidine (TMB, Sigma) colorimétrie development was then measured by a Tecan Safire2 (Austria, Europe) microplate reader at 450 nm.

Plasma Factor Vil (F7) activity measurements. Plasma samples from mice were prepared by collecting blood (9 volumes) by submandibular bleeding into microcentrifuge tubes containing 0.109 mol/L sodium citrate anticoagulant (1 volume) following standard procedures. F7 activity In plasma is measured with a chromogenic method using a BIOPHEN Vil kit (Hyphen BioMed/Aniara, Mason, OH) following manufacturées recommendations. Absorbance of colorimétrie development was measured using a Tecan Safire2 microplate reader at 405 nm.

Example 7. Delivery of siRNA to hépatocytes in vivo using siRNA-hydrophobe conjugates co· administered with masked DW1360 delivery polymer. siRNA and delivery poiymer were prepared and administered as described above using the indicated doses of siRNA and polymer.

RNAI polynucleotide delivery to hépatocytes In vivo. Co-administration of siRNA-cholesterol conjugale and masked DW1360 delivery polymer resulted In decreased sérum ApoB protein levels, indicating delivery of the siRNA to hépatocytes and inhibition of apoB gene expression. Efficient delivery required both the delivery polymer and cholestérol conjugation to the RNAi poiynucieotide (Table 1, FIG. 2). No significant knockdown was observed with up to 5 mg/kg unconjugated siRNA. Further, the hydrophobie group could be attached to either the 5’ or 3' end of the siRNA,

Table 1. Knockdown of target gene in vivo following Injection of siRNA-hydrophobe conjugate plus DW1360 delivery poiymer, effect of siRNA-conjugate dose.

siRNA	siRNA dose (mg/kg)	Poiymer dose (mg/kg)	Relative % ApoB*b
5' cholestérol apoB	0.1	20	75 ±5
0.25	20	42 ±3
0.5	20	2516
1	20	26117
3' cholestérol apoB	1	20	25 12
5	0	102 133
unconjugated siRNA	0.5	16	8714
5	16	71 120

* Percent knockdown relative to control group (n=3) Injected with isotonie glucose solution.

^b ICR mice

B. Effect of hydrophobie group size on RNA! poiynucieotide delivery to hépatocytes. Efficient delivery of siRNA to hépatocytes, using co-administration with DW1360 delivery poiymer 10 required that the siRNA be conjugated to a hydrophobie group having about 20 or more carbon atoms (Table 2, FIG. 3). siRNA-hydrophobe conjugates having hydrophobie targeting molettes with fewer than 20 carbon atoms exhibited progressively less efficient functional delivery.

Hydrophobe targeting molettes having six (6) or fewer carbons were Ineffective. Delivery efficlency was not significantly improved by increasing the size of the hydrophobe targeting 15 molety beyond 20 carbon atoms.

Table 2. Knockdown of target gene in vivo following Injection of siRNA-hydrophobe conjugate plus DW1360 delivery poiymer - effect of hydrophobie group size.

SiRNA

Carbon atomsb

siRNA dose (mg/kg)

Poiymer dose (mg/kg)

Relative % Factor VII *e

5'-hexyl fVII	6	2.5	12.5	108 ±18
S'-dodecyl fVII	12	2.5	12.5	66 ±18
5-octadecyl fVII	18	2.5	12.5	61 ±19
5’-(decyl)2 fVII	20	2.5	12.5	31 ±8
5'-(dodecyl)2 fVII	24	2.5	12.5	15 ±5
5'-cholesterol fVII	27	2.5	12.5	15 ±3
5'-(octadecyl)2 fVII	36	2.5	12.5	16 ±3

* Percent knockdown relative to controi group (n=3) Injected with isotonie glucose solution. ^b number of carbon atoms in the hydrophobie group conjugated to the siRNA ^e C57BL/6 mice

C. Effect of siRNA dose on siRNA-hydrophobe conjugale delivery to hépatocytes. Knockdown of target gene expression In vivo Is dépendent on siRNA dose. For treatment of mice, administration of more than 1.25 mg/kg siRNA dose did not improve target gene knockdown In vivo (Table 3, FIG. 4). Dosage as low as 0.25 mg/kg did however provide significant knockdown of target gene expression in mice when co-administered with delivery polymer.

Table 3. Knockdown of target gene in vivo following injection of siRNA-hydrophobe conjugate plus DW1360 delivery polymer - effect of siRNA dose.

SiRNA	siRNA dose (mg/kg)	Polymer dose (mg/kg)	Relative % Factor VII ·“
5-(dodecyl)2 fVII	2.5	12.5	15 ±5
1.25	12.5	25 ±6
0.5	12.5	44 ±14
0.25	12.5	61 ±8
5-(octadecyl)2 fVl I	2.5	12.5	16 ±3
1.25	12.5	12 ±9
0.5	12.5	23 ±10
0.25	12.5	25 ±3
5'-cholesteroi fVII	2.5	12.5	15 ±3
1.25	12.5	9±1
0.5	12.5	31 ±8
0.25	12.5	28 ±11

• Percent knockdown relative to control group (n=3) injected with isotonie glucose solution. ^b C57BL/6 mice

D. Knockdown of target gene expression in vivo Is dépendent on delivery polymer dose. For treatment ot mice, administration of about 12,5 mg/kg delivery polymer provided maximal or near maximal RNAI-polynucleotide delivery as evidenced by target gene inhibition (Table 4, FIG. 5). Knockdown of target gene is affected by polymer dose. Excess siRNA-conjugate did not improve target gene knockdown in the absence of sufficient polymer for delivery.

Table 4. Knockdown of target gene In vivo following injection of siRNA-hydrophobe conjugale plus DW1360 delivery polymer - effect of delivery polymer dose.

siRNA	siRNA dose (mg/kg)
3'-cholesterol apoB	1
1
1
1
1
1
1
1
10
10

Polymer dose (mg/kg)	Relative % ApoB*
5	112 ±11b
8.75	54±20b
12.5	27±5*
17.5	28 ±14b
25	12 ±4“
3.75	91 ±21c
7	59±30e
12.5	38±19e
3.75	74 ±13e
7	71 ±24c

¹ Percent knockdown relative to control group (n=3) injected with isotonie glucose solution.

^b ICR mice ^c C57BL/6 mice

E. Sequential administration. The RNAi polynucleotide-hydrophobe targeting moiety conjugate and delivery polymer may be administered to the animal sequentially. For RNAi polynucleotidehydrophobic targeting moiety conjugates, the RNAI conjugate may be administered up to 30 minutes prior to administration of the delivery polymer. Also for RNAi polynucleotidehydrophobie targeting moiety conjugates, the delivery polymer may be administered up to two hours prior to administration of the RNAi conjugate (Table 5).

Table 5. Knockdown of target gene in vivo followlng injection of siRNAhydrophobe conjugate plus DW1360 delivery polymer - effect of sequential administration of siRNA and polymer.

siRNA	First Injection	Intervai	Second Injection	Relative % ApoB*
5-cholesterol apoB	0.5 mg/kg siRNA	15 min	12.5 mg/kg polymer	25 ±5
30 min	35 ±13
120 min	90 ±20
12.5 mg/kg polymer	120 min	0.5 mg/kg siRNA	20 ±5
3'-cholesterol apoB	0.5 mg/kg siRNA	0 min	12.5 mg/kg polymer	27 ±11
15 min	25 ±9
30 min	34 ±12
12.5 mg/kg polymer	15 min	0.5 mg/kg siRNA	41 ±6
30 min	41 ±15

• Percent protein relative to control group (n=3) Injected with Isotonie glucose solution.

F. Membrane active poly(acrylate) delivery polymers. Reversibly masked amphipathlc membrane active poly(acrylate) polyamines fonction as effective delivery polymers.

Poly(acrylate) polymers were prepared as described above and co-administered with siRNAcholesteroi conjugates In mice as described for DW1360 delivery polymers. The poly(acrylate) delivery polymers were effective in facilitating delivery of siRNA to hépatocytes in vivo as indicated by reduced sérum ApoB (Table 6). Efficient delivery required both the delivery polymer and cholestérol conjugation to the RNAi polynucleotide.

Table 6. Knockdown of target gene in vivo following injection of siRNA-hydrophobe conjugate plus masked poly(acrylate) delivery polymers.

Poly(acrylate) polymer	siRNA	siRNA dose (mg/kg)	Polymer dose (mg/kg)	Relative % ApoB
Ant 40911-3 23- 38	5' cholestérol apoB	0.5	15	14 ±4

Ant 40911-35-2

5' cholestérol apoB

0.5

20

23 ±10

G. Delivery of RNAI polynucleotide-hydrophobe conjugate to liver was not dépendent on either the LDL-Receptor or the LJpoprotein Receptor-Related Protein. Co-administration of Factor VII siRNA-cholesterol conjugate and masked DW1360 delivery poiymer resulted In decreased in 5 sérum Factor VII protein levels in LDL-Receptor knockout mice and Lipoprotein ReceptorRelated Protein/LDL-Receptor double knockout mice. Therefore, siRNA-cholesterol is targeted to hépatocytes by means other than LDL particles, LDL-Receptor or Lipoprotein ReceptorRelated Protein (Table 7).

Table 7. Knockdown of target gene In vivo following injection of siRNAcholesterol conjugate plus DW1360 delivery poiymer; effect of LDL receptor and Lipoprotein Receptor-Related Protein on siRNA delivery.

siRNA	siRNA dose (P9)	Poiymer dose* (P9)	Poiymer modification	Relative % Factor VII
LDL Receptor knockou	tmice
cholesterolsiRNA Factor VII	0	0		100 ±18
20	400	NAG + PEG	35 ±18
20	400	NAG	26 ±7
20	400	PEG	99 ±9
LJpoprotein Receptor-Related Protein / LDL-Receptor double knockout mice
cholesterolsiRNA Factor VII	0	0		100 ±20
20	400	NAG + PEG	11 ±4
20	400	NAG	26 ±9
20	400	PEG	88 ±26

' relative % protein

H. Lyophilized polyfvinyl ether) samples. To test whether the delivery poiymer could be lyophilized for Improved storage and transport, delivery poiymer In solution was frozen and placed In high vacuum on a lyophllizer. After 16 h, the sample was a crystalline powder that 15 was then redissolved by addition of deionized water. To the redissolved poiymer sample was added siRNA (5'cholesterol apoB), and the sample was Injected. Lyophllization showed no detrimental effects on the delivery polymer.

Galactose Cluster Targeted siRNA

Example 8. Delivery of siRNA to hépatocytes In vivo using siRNA-galactose cluster conjugates co-adminlstered with masked DW1360 delivery polymer. siRNA and delivery polymer were prepared and administered as described above using the Indicated doses of siRNA and . polymer.

Co-admlnlstration of siRNA-galactose cluster conjugate and masked DW1360 delivery polymer. Co-administration of siRNA-galactose cluster conjugate and masked DW1360 delivery polymer resulted in decreased sérum ApoB protein levels, indicating delivery of the siRNA to hépatocytes and Inhibition of apoB gene expression. Efficient delivery required both the delivery polymer and galactose cluster conjugation to the RNAi polynucleotide (Table 8). No slgnificant knockdown was observed with up to 5 mg/kg unconjugated siRNA. As with the hydrophobe conjugate siRNA above, onset of maximum inhibition is obtained with about 12.5 mg/kg delivery polymer dose. No target gene knockdown was observed In the absence of co-administered delivery polymer. The galactose ciuster-siRNA conjugate exhibited no activity by itself.

Table 8. Knockdown of target gene in vivo followlng injection of siRNA-GalNAc cluster conjugate plus delivery polymer, effect of polymer dose.

siRNA	siRNA dose a (mg/kg)	Polymer dose1 (mg/kg)	Relative % ApoBb
5'GaiNAc cluster apoB	0.5	10	48 ±9
0.5	20	26 ±12
0.5	40	15 ±6
0.5	60	18±10
unconjugated siRNA	0.5	16	87 ±4

• mg siRNA or polymer per kilogram anima weight ^b relative % protein

B. siRNA-galactose cluster vs. siRNA-galactose monomer. Functionat delivery of siRNA to hépatocytes In vivo when co-administered with delivery polymer required a tri-antennary galactose targeting moiety conjugated to the RNAI interférence polynucleotide. No target gene knockdown was observed when a single galactose molecuie was conjugated to the siRNA (Table 9). The GaINPr (N-propionyl galactosamine) galactose dérivative is known to hâve a higher affinity for the ASGPr than the GalNAc (N-acetyl-galactosamlne) galactose dérivative, further indicating the necessity of the triantennary galactose cluster for efficient delivery.

Table 9. Knockdown of target gene In vivo following injection of siRNA-GalNAc cluster conjugate plus delivery polymer; trivalent vs. monovalent galactose RNA conjugate.

siRNA	Ligand	siRNA dose* (mg/kg)	Polymer dose* (mg/kg)	Relative % proteinb
apoB	5'GalNPr monomere	0.25	12.5	100 ±7
5'GalNAc cluster d	0.25	12.5	56 ±11

• mg siRNA or polymer per kilogram animal weight ^b relative % proteln ^c N-Propionyl Galactosamine monomer ^d N-Acetyl Galactosamine cluster (trimer)

C. Effect of modification of polymer with galactose dérivative, PEG, or galactose dérivative plus PEG. siRNA-galactose cluster and delivery polymer were prepared as described above except as follows: the delivery polymer was either masked with N-acetylgalactosamlne alone, PEG alone, or N-acetylgalactosamine plus PEG. siRNA and delivery polymer were then administered to mice as described above. Blood samples were then collected from mice and assayed to détermine ApoB levels. Both galactose and PEG were required for optimal delivery. By modifying the membrane active polymer with both galactose and PEG, only half of the siRNA dose was required to achieve the same effect and polymer modified with galactose alone. Modification of polymer with PEG alone resulted in decreased siRNA delivery compared to polymer modified with galactose alone or with galactose plus PEG.

Table 10. Knockdown of target gene in vivo following injection of siRNAGalNAc cluster conjugale plus delivery polymer, effect of polymer modification.

siRNA	siRNA dose' (mg/kg)	Polymer dose (mg/kg)	Polymer modification	Relative % ApoB6
5'GalNAc cluster apoB	0.5	20	CDM-NAG + CDM-PEG	26 ±12
1	20	CDM-NAG	23 ±10
1	20	CDM-PEG	45 ±10

* mg siRNA or polymer per kilogram animal weight ⁶ relative % protein

D. Time course of sequence spécifie gene knockdown following co-administration of siRNA5 targeting moiety conjugale and delivery polymer. siRNA and delivery polymer were prepared as and adminlstered to mice as described above. Blood samples were then collected from mice at the Indicated times and assayed to détermine ApoB levels. ApoB ievels were observed to graduaily decrease until they reached 3% of control lever after 72 h. Thus, maximum target gene knockdown may occur after about three (3) days. This delay in onset of maximum 10 decrease in protein leveis may reflect the time required to clear or dégradé ApoB protein rather than the time requlred for maximum RNAi polynucleotide delivery or for gene knockdown.

Table 11. Knockdown of target gene in vivo foliowing injection of siRNAGalNAc cluster conjugale plus delivery polymer; time course of target gene knockdown.

SiRNA	siRNA dose1 (mg/kg)	Polymer dose* (mg/kg)	Hours post injection	Relative % protein6
5’GalNAc cluster apoB	1	20	5	134 ±15
24	35 ±3
48	12 ±2
72	3±1

* mg siRNA or polymer per kilogram animal weight ⁶ relative % protein

E. Sequential Injection of siRNA-galactose cluster conjugale and delivery polymer. The Indicated amounts of siRNA-galactose cluster conjugate and delivery polymer were prepared and administered to mice as described above. Blood samples were then collected from mice and assayed to détermine ApoB levels. For siRNA targeted to the liver with the galactose 5 cluster, optimal delivery was observed with simultaneous delivery of the siRNA and delivery polymer. Significant siRNA delivery was observed when the siRNA-conjugate was administered up to 15 minutes after administration of the polymer. Only modest delivery was observed when the siRNA-conjugate was administered prior to (up to 15 minutes) the delivery polymer.

Table 12. Knockdown of target gene in vivo foliowing injection of siRNAGalNAc cluster conjugate plus delivery polymer; simultaneous administration and separate administration.

siRNA	First injection	Interval	Second injection	Relative % apoB
5'GalNAc cluster apoB	0.25 mg/kg siRNA	0 min	12.5 mg/kg polymer	28 ±14
12.5 mg/kg polymer	15 min	0.25 mg/kg siRNA	56 ±18
0.25 mg/kg siRNA	15 min	12.5 mg/kg polymer	88 ±14

F. Insertion of a PEG lînker between the galactose cluster targeting ligand and the RNAi polynucleotide. siRNA-galactose cluster conjugates were either prepared inserting PEG spacers, PEG₁₉ or PEG₂«, between the galactose cluster and the siRNA or prepared without a PEG spacer between the galactose cluster and the siRNA. The siRNA-conjugates were then 15 co-administered with delivery polymer. Insertion of PEG spacers did not improve delivery of the siRNA to hépatocytes as determined by gene knockdown.

Galactose cluster without PEG spacer; targeting ligand attached to the siRNA through the carboxyl group.

Galactose cluster with PEG spacer; targeting ligand attached to the siRNA through the carboxyl group.

Table 13. Knockdown of target gene in vivo following injection of siRNAGalNAc cluster conjugate plus delivery polymer; effect of PEG linker in RNA conjugate.

SiRNA	siRNA dose * (mg/kg)	PEG linker	Polymer dose * (mg/kg)	Relative % ApoBb
5'GalNAc cluster apoB	0.25	none	12.5	28 ±14
5'GalNAc cluster-PEG1B apoB	0.25	PEG1S	12.5	82 ±19
5’GalNAc cluster-PEG24 apoB	0.25	peg2<	12.5	72 ±13

‘ mg siRNA or polymer per kilogram animal weight ^b relative % protein

Example 9. Delivery of siRNA to primate hépatocytes in vivo. RNAi polynucleotide conjugates and masked polymers were synthesized as described above.

A Rhésus monkey (3.9 kg male) was injected I.V. with 7.8 mL of a solution containing 1.0 mg/ml cholesterol-slApoB and 7.5 mg/ml DW1360 modified with 7^X wt ratio of 2:1 CDM-PEG:CDMNAG, givlng a final dose of 2 mg/kg cholesterol-siApoB and 15 mg/kg DW1360. Another Rhésus monkey (4.5 kg male) was injected with Isotonie glucose and served as a control.

Sérum ApoB levels détermination. Sérum ApoB proteln levels were monitored during the course. Primates was fasted for 4 h before sérum collection. Sérum ApoB protein levels were determined by standard sandwich ELISA methods. Briefly, a polyclonal goat anti-mouse ApoB antibody and a rabbit anti-mouse ApoB antibody (Biodesign International) were used as capture 10 and détection antibodies respectively. An HRP-conjugated goat anti-rabbit IgG antibody (Sigma) was applied afterwards to bind the ApoB/antibody complex. Absorbance of tetramethylbenzidine (TMB, Sigma) colorimétrie development was then measured by a Tecan Safire2 (Austria, Europe) microplate reader at 450 nm. The results are given in Table 14. The Rhésus monkey receiving the cholesterol-slApoB siRNA showed a decrease in sérum ApoB levels over 15 time, reaching a maximum knockdown of 76% on Day 15 after Injection compared to Day -1 pre-dose levels. ApoB levels recovered to the near Day -1 pre-dose levels on Day 50. No decrease in sérum ApoB levels were observed In the control animal.

Table 14. Sérum ApoB levels normalized to Day 1.

Day	Treatment
Isotonie glucose	chol-siRNA (ApoB) + polymer
1	1.00	1
2	1.24	1.07
4	1.38	0.69
7	1.22	0.56
11	1.39	0.32
15	1.43	0.24
18	1.36	0.25
22	1.44	0.31
29	1.13	0.30
36	1.21	0.48

Example 10. Simultaneous knockdown of two genes. Co-administration of siRNA-cholesterol conjugales to two Independent genes, apoB and factor Vil, and masked DW1360 delivery polymer resulted In simultaneous Inhibition of both genes. The composition was admlnistered to mlce as described above. (Table 15).

Table 15. Simultaneous knockdown of 2 target genes In vivo followlng Injection of two different siRNA-hydrophobe conjugales plus 400 pg DW1360 delivery polymer.

3' cholesterolapoB (pg)	3’ cholesterolfactor VII (pg)	Relative % ApoB*	Relative % Factor VII*
0	0	100 ±19	100 ±25
20	0	12 ±4	124 ±21
0	20	81 ±12	14 ±5
20	20	10 ±6	12 ±1

* Percent knockdown relative to control group (n=3) injected with Isotonie glucose solution.

Toxicity Evaluation

Example 11. Toxicity. The potential toxicity of the delivery System was assessed by measuring sérum levels of liver enzymes and cytokines. Slight élévations of ALT and AST levels were detected in mice receiving control siRNA or apoB-1 slRNA conjugates as compared to salinetreated mice 48 h after injection. However, the Increased levels were not significant (p<0.05), and histological examination of liver sections did not reveal signs of liver toxicity. Similarly, analysis of TNF-α and IL-6 levels in sérum uslng ELISA revealed that both were slightly elevated 6 h after injection of siRNA-polymer conjugate. The levels of both retumed to baseline by 48 h. No statistically significant toxicity was measured at the minimal effective dose in mice or rats. These results indicate the targeted delivery System was well-tolerated.

Example 12. The siRNAs had the following sequences:

apoB siRNA:

sense 5' GGAAUCuuAuAuuuGAUCcAsA 3' (SEQ ID 1) antlsense 5' uuGGAUcAAAuAuAAGAuUCcscsU 3' (SEQ ID 2) factor VII siRNA sense 5' GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdT 3' (SEQ ID 3) antisense 5’ GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT 3' (SEQ ID 4) small letter ® 2'-O-CH₃ substitution s = phosphorothioate linkage f after nucléotide = 2'-F substitution d before nucléotide = 2'-deoxy

Example 13. Synthesis ofGalNAc cluster.

{2-[2-(2-Hydmxy-ethoxy)-ethoxy]-ethoxy}-acetic acid benzyl ester

2-[2-(2-Hydroxy-ethoxy)-ethoxy]-ethanoi (62.2 g, 414 mmol) was dissolved under argon In 875 mL of abs. DMF and cooled to O’C. NaH (12.1 g. 277 mmol, 55 % In minerai oil) was carefully added, the ice bath removed, and stirring continued for 1 h at 80’C. The reaction mixture was 10 cooled to ambient température and treated with bromoacetic acid (18.98 g. 137 mmol) which was added via dropplng funnel as a DMF-solution (20 ml). After an additional 30 min. at 75’C, bromomethyl-benzene (23.36 g, 137 mmol) was added neat and estérification allowed to proceed for 30 min. Cooling, careful pouring onto crashed ice, extraction with ethyl acetate, washing with water, drying over Na₂SO₄, and évaporation of ali solvents followed by flash 15 chromatography (SIO₂, ethyl acetate / heptane = 8/2) yielded 6.41 g of the title compound as a yellow oil. MS (ISP): 299.2 [M+H]*.

B. Acetic acid (3aR,5R,6R.7R,7aR)-6-acetoxy-5-acetoxymethyl-2-methyl-5,6_i7,7a-tetrahydro-

3aH~pyrano[3,2-d]oxazol-7-yl ester.

Commercially available acetic acid (2S,3R,4R,5R,6R)-4,5-diacetoxy-6-acetoxymethyl-3acetylamino-tetrahydro-pyran-2-yl ester (10.0 g, 26 mmol) was dissolved In 116 mL of abs. CH₂CI₂ and treated with trimethylsilyl triflate (14.27 g, 64 mmol). The reaction was allowed to proceed over night at 45*C. After cooling to O’C, triethylamine (4.88 ml, 35 mmol) was added, the mixture diluted with CH₂CI₂ and washed with NaHCO_rsolution and water. Drying over Na₂SO₄ and évaporation of the solvent yielded 10.3 g of the title compound as brownlsh oil which was used without further purification for the next step. MS (ISP): 330.0 [M+H]*.

C. (2-{2-[2-((2R, 3R, 4R, 5R, 6R)-4,5-Diacetoxy-6-acetoxymethyl-3-acetylamino-tetrahydro-pyran2-yloxy)-ethoxy]-ethoxy}-ethoxy)-ecetic add benzyl ester.

Chiral

O

The above prepared acetic acid (3aR,5R,6R,7R,7aR)-6-acetoxy-5-acetoxymethyl-2-methyl5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-7-yl ester (10.3 g, 26 mmol) and {2-[2-(2-hydroxy· ethoxy)-ethoxy]-ethoxy}-acetic acid benzyl ester (8.62 g, 29 mmol) were mlxed In 520 mL of CH₂CI₂ and treated with 63 g of 4 Angstrom molecular sieves. After 1 h trimethylsilyl triflate (6.13 g, 28 mmol) was added. The réaction mixture was stirred over the weekend at ambient température. Triethylamlne (5.21 ml, 37 mmol) was added, the molecular sieves filtered off, the filtrate diluted with CH₂CI₂ and washed with NaHCO₃-solution and water. Drying over Na₂SO₄ and évaporation of the solvent followed by flash chromatography (SiO₂, ethyl acetate / AcOH / MeOH / water = 60/3/3/2) afforded 15.7 g of the title compound as a brownish oil. MS (ISP): 626.6 [M-H]·.

D. (2-{2-[2-((2R,3R,4R,5R,6R)~4,5-Diacetoxy-6-acetoxymeth^‘3-acetylamlno-tetrahydro-pyran2-yloxy)-ethoxyJ-ethoxy}-ethoxy)-aceticadd.

O Chiral

O

O

The above prepared (2-{2-[2-((2R,3R,4R,5R,6R)-4,5-diacetoxy-6-acetoxymethyl-3-acetylaminotetrahydro-pyran-2-yloxy)-ethoxy]-ethoxy}-ethoxy)-acetic acid benzyl ester (15.7 g, 25 mmol) was dissolved In 525 mL of ethyl acetate and hydrogenated over 1.6 g of Pd / C (10%) under 1 atm. of H₂ at ambient température for 3 h. Filtration over Celite and évaporation of the solvent, followed by flash chromatography (S1O2, CH2CI2 / MeOH ^e 80 / 20) gave 6.07 g of the title compound as a brownish gum. MS (ISP): 536.5 [M-Hf.

The above prepared (2-{2-[2-((2R,3R,4R,5R,6R)-4,5-diacetoxy-6-acetoxymethyl-3-acetylaminotetrahydro-pyran-2-yloxy)-ethoxy]-ethoxy}-ethoxy)-acetic acid (2.820 g, 5.246 mmol) and (S)-6amino-2-((S)-2,6-diamino-hexanoylamino)-hexanolc acid benzyl ester hydrochloride (préparation see below, 0.829 g, 1.749 mmol) were dissolved in a mixture of 32 mL of CH₂CI₂ and 3.2 mL of DMF, treated successively with Hünïg’s base (2.096 ml, 12.25 mmol), 1-hydroxy7-azabenzotriazole (0.714 g, 5.248 mmol) and 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochloride (1.006 g, 5.248 mmol), and stirred over night at ambient température. Ail volatiles were removed i.V., and the crude reaction mixture purified by préparative HPLC (38 runs,

Gemini, 5D, C18) to give after lyophilization 1.650 g of the title product as white powder. MS (ISP): 1945.8 (M+NaJ*. NMR (600 MHz, DMSO).

F. GalNAc Cluster free acid. (17S,20S)-1-((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6' (acetoxymethyl)tetrahydro-2H-pyran-2-yloxy)-20-(1-((2R,3R,4R,5R,6R)-3-acetamido-4,5diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yloxy)-11-oxo-3,6,9-trioxa-125 azahexadecan-16-yl)-17-(2-(2-(2-(2-((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6(acetoxymethyl)tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)ethoxy)acetamido)-11,18-dioxo3,6,9-trioxa-12,19-diazahenicosan-21-oicacid.

The above prepared GalNAc Cluster benzyl ester (0.674 g, 0.350 mmol) was dissolved in 50 mL of MeOH and hydrogenated over 0.065 g of Pd / C (10%) under 1 atm. of H₂ at ambient température for 4 h. Filtration over Celite and évaporation of the solvent left 0.620 g of the title compound as a white foam. MS (ISP): 1917.0 [M+2H]²*. NMR (600 MHz, DMSO).

Example 14. (S)-6-amino-2-((S)-2,6-diamino-hexanoylamino)-hexanoic acid benzyl ester hydrochloride. The building block (S)-6-amino-2-((S)-2,6-diamino-hexanoylamino)-hexanoic acid benzyl ester hydrochloride was syntheslzed as follows:

(S)-6-tert-Butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamlno)· hexanolc acid benzyl ester.

(S)-6-tert-Butoxycarbonylamino-2-(9H-fluoren-9-yimethoxycarbonylamino)-hexanoic acid (5.00 g, 10.67 mmol) and phenyl-méthanol (2.305 g, 21.34 mmol) were dissolved in 25 mL of CH₂CI₂ and treated successively with N-hydroxybenzotriazole (1.933 g, 11.74 mmol), 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC, 2.250 g, 11.74 mmol), and ethyldiisopropyl-amine (2.137 ml, 12.49 mmol). After stirring for 90 min, the volatiles were removed I.V. at ambient température, the residue taken up In ethyl acetate, washed with water, NH₄CIsolution and brine, dried over Na₂SO₄, and evaporated. The crude mixture was then dissolved in 20 mL of éthanol, and the product precipitated by adding 10 mL of water. Filtration and drying *

yielded 5.669 g of the title compound which was recrystallized from éthanol / hexane to give 4.27 g of pure benzyl ester. MS (ISP): 559.2 [M+H]*.

B. (S)-2-((S)-2,6-Bis-tert-butoxycarbonylamlno-hexanoylamino)~6-tert-butoxycarbonylaminohexanolc acid benzyl ester.

Chiral

The above prepared (S)-6-tert-Butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoic acid benzyl ester (4.270 g, 7.643 mmol) was dissolved In 15 mL of THF and treated with 15 mL of diethyiamlne. After 4 h at ambient température MS and TLC indicated the absence of starting matériel. Evaporation of the solvents and azeotroplc drylng with toiuene afforded 4.02 g of the free amine which was used directly In the next step.

Commercially available (S)-2,6-bis-tert-butoxycarbonylamino-hexanoic acid (3.177 g, 9.17 mmol) was dissolved In 13 mL of CH₂CI₂ and treated at 0’C with ethyi-diisopropyl-amine (4.71 ml, 27.5 mmol), O-(1,2-dihydro-2-oxo-pyridyl)- -1,1,3,3-tetramethyluronlum tetrafluoroborate (TPTU, 2.725 g, 9.172 mmol) and, 15 min. iater, with the above prepared amine as a solution In minimal CH₂CI₂ and 1.57 mL of ethyi-diisopropyl-amine (1,2 eq.). The reaction was allowed to proceed for 2 h at ambient température. Ail volatiles were removed I.V., the residue taken up in ethyi acetate, washed with NaHCOj-solution, NH₄CI-soiution and water, dried over Na₂SO₄, and evaporated. Flash chromatography (SiO₂, heptane / ethyi acetate - 4 / 6) followed by crystailization from heptane / minimal amounts of ethyi acetate produced 4.516 g of the title compound as a white solid. MS (ISP): 665.4 [M+H]*.

C. (S)-6-Amino-2-((S)-2,6-diaminohexanoylamino)-hexanoic acid benzyl ester trihydrochloride.

The above prepared (S)-2-((S)-2,6-bls-tert-butoxycarbonylamlno-hexanoylamino)-6-tertbutoxycarbonyiamino-hexanoic acid benzyl ester (4.516, 6.793 mmol) was dissolved In 4 mol/L HCl In dioxane. After a couple of minutes, gas evolved and a precipitate was formed. After 3 h at ambient température, the reaction mixture was carefully evaporated and scrupulously dried to yield 3.81 g of the title compound as an off-white foam which was used without further purification for Example 13. E. GalNAc Cluster benzyl ester above. MS (ISP): 365.3 [M+H]*.

Example 15. GalNAc cluster-siRNA conjugates.

Compound 1 (150 mg, 0.082 mmol) was dissolved In dry methanol (5.5 ml) and 42 pL sodium methylate were added (25% solution in MeOH). The mixture was stirred under an argon atmosphère for 2 h at RT. An equal amount of methanol 5 was added as well as portions of an anionic exchange matériel Amberlite IR120 to generate a pH around 7.0. The Amberlite was removed by filtration. The solution was dried with Na₂SO4, and the solvent was removed under reduced pressure. Compound 2 was obtained in quantitative yield as a white foam. TLC (SiO₂, dichloromethane (DCM)/MeOH 5:1 + 0.1% CH₃COOH): R, 2 = 0.03; for 10 détection a solution of suifuric acid (5%) in MeOH was used followed by heating. ES1-MS, direct injection, négative mode; (Μ-Η]·¹^.»^ 1452.7; [M-H]¹*^.,^: 1452.5.

Compound 2 (20 mg, 0.014 mmol) was co-evaporated with pyridine and 15 dichloromethane. The residue was dissolved in dry DMF (0.9 ml) and a solution of N-Hydroxysuccinimide (NHS) in DMF (1.6 mg, 0.014 mmol) was added while stirring under an argon atmosphère. At 0°C a solution of N,N'Dicyclohexylcarbodiimide (DCC) in DMF (3.2 mg, 0.016 mmol) was slowly added. The reaction was allowed to warm to RT and stirred over night.

Compound 3 was used without further purification for conjugation to RNA.

Synthesis of amino-modified RNA. RNA equipped with a C-6-amino linker at the 5-end of the sense strand was produced by standard phosphoramidite chemistry on solid phase at a scale of 1215 pmol using an ÀKTA Oligopilot 100 25 (GE Healthcare, Freiburg, Germany) and controlled pore glass as solid support.

RNA containing 2'-O-methyl nucléotides were generated employing the corresponding phosphoramidites, 2'-O-methyl phosphoramidites and TFAhexylaminolinker amidite. Cleavage and deprotection as weil as purification was achieved by methods known in the field (Wincott F., et al. NAR 1995, 30 23,14,2677-84).

The amino-modified RNA was characterized by anion exchange HPLC (purity: 96.1%) and identity was confirmed by ESI-MS ([M+H]¹* 6937.4; [M+H] ¹*™„_Ur»d: 6939.0. Sequence:

5'-(NH₂C_e)GGAAUCuuAuAuuuGAUCcAsA-3' (SEQ ID 1); u,c: 2'-O-methyl nucléotides of 35 corresponding bases, s: phosphorothioate.

D. Conjugation of GalNAc Cluster to RNA. RNA (2.54 pmol) equipped with a C-6 amlno linker at the 5'-end was lyophilized and dissolved In 250 μΙ_ sodium borate butter (0.1 mol/L sodium borate, pH 8.5, 0.1 mol/L KCI) and 1.1 mL DMSO. After addition of 8 pL Ν,/V-Diisopropylethylamlne (DIPEA), a solution of compound 3 (theoretically 0.014 mmol) in

DMF was slowly added under continuous stirring to the RNA solution. The reaction mixture was agitated at 35’C ovemight. The reaction was monitored using RP-HPLC (Resource RPC 3 ml, butter: A: 100 mM Triethylammonium acetate (TEAA, 2.0 M, pH 7.0) in water, B: 100 mM TEAA In 95% acetonitrile, gradient: 5% B to 22% B In 20 CV). After précipitation of RNA using sodium acetate (3 M) In EtOH at -20’C, the RNA conjugate was purified using the conditions described 10 above. The pure fractions were pooled, and the desired conjugate 4 was precipitated using sodium acetate/EtOH to give the pure RNA conjugate. Conjugate 4 has been isolated in 59 % yield (1.50 pmol). The purity of conjugate 4 was analyzed by anion exchange HPLC (purity: 85.5 %) and Identity was confirmed by ESI-MS ([M+H]¹* 8374.4; [Μ+Η]Ά„_υΓ^: 8376.0.

(FIG. 6.)

E. Conjugate 4 (sense strand) was annealed with an 2-O-methyl-modified antisense strand. Sequence: 5-uuGGAUcAAAuAuAAGAuUCcscsU-3' (SEQ ID 2). The siRNA conjugate directed against the apolipoprotein B mRNA was generated by mixing an equimolar solution of complementary strands in annealing butter (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90’C for 3 min, and cooled to RT over a period of 3-4 h. Duplex formation was confirmed by native gel electrophoresis.

Example 16. Hydrophobie group-siRNA conjugates.

SEQ ID 3:

(NHSCIO)GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTsdT

Amine (Amine)(COC9)GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTsdT

RNA synthesls was performed on solid phase by conventional phosphoramidite chemistry on an ÀKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany) and controlled pore glass (CPG) as solid support.

The 5-C10-NHS ester modified sense strand, (NHSCIO)GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTsdT (SEQ ID 3) was prepared employing 5'-Carboxy-Modifier C10 amidite from Glen

Research (Virginia, USA). The activated RNA, still attached to the solid support was used for conjugation with lipophilie amines listed In the table below. Cf and Uf are 2'-fIuoronucleotides of the corresponding bases and s is a phosphorothioate linkage.

Sense strand sequence: 5'-(COC9)GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTsdT-3’ (SEQ ID 3) Antisense strand sequence: 5'-GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT-3' (SEQ ID 4)

100 mg of the sense strand CPG (loading 60 pmol/g, 0.6 pmol RNA) were mixed with 0.25 mmol of the corresponding amine obtained from, Sigma Aldrich Chemie GmbH (Taufklrchen, 10 Germany) or Fluka (Sigma-Aldrich, Buchs, Switzerland).

Table 16. Lipophilie amines used In forming hydrophobie group-siRNA conjugates

N r	Lipophilie Amine	m 9	mm ol	ml solvent
2	N-Hexylamine	25	0.25	1 mL CH2CI2
3	Dodecylamine	50	0.25	0.55 mL CH3CN, 0.45 mL CH2CI2
4	Octadecylamin e	67	0.25	1 mLCH2CI2
5	Didecylamine	74	0.25	1 mL CH2CI2
6	Didodecylamine	88	0.25	1 mL CH2CI2
7	Dioctadecylami ne	67	0.12	0.45 mL CH2CI2, 0.45 mL Cyclohexane

The mixture was shaken for 18 h at 40’C. The RNA was cleaved from the solid support and 15 deprotected with an aqueous ammonium hydroxlde solution (NH₃, 33 %) at 45’C overnight. The 2'-protecting group was removed with TEAx3HF at 65’C for 3.5 h. The crude oligoribonucleotides were purified by RP-HPLC (Resource RPC 3 ml, buffer: A: 100 mM TEAA in water, B: 100 mM TEAA In 95% CH₃CN, gradient.^- 3% B to 70% B in 15 CV, except for Nr 7 : gradient from 3% B to 100% B In 15 CV).

Table 17. Hydrophobie group-RNA conjugates, characterized by RP- HPLC and ESI-MS (négative mode).

Nr	Purity RPHPLC %	ESI-MS [M-H] calculated	ESI-MS [M-H] found
2	90	6963.4	6963.0

3	99	7047.4	7047.2
4	98	7131.5	7131.4
5	99	7159.6	7159.3
6	99	7215.7	7215.0
7	98	7384.0	7383.2

To generate siRNA from RNA single strand, equimolar amounts of complementary sense and antisense strands were mixed in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated at 80^eC for 3 min, and cooled to RT over a period of 3-4 h. The siRNA, 5 which are directed agalnst factor VII mRNA were characterized by gel electrophoresis.

Claims (30)

1. CLAIMS:

   1. A composition for delivering an oligonucleotide to a liver cell in vivo comprising:

   a) an oligonucleotide covalently linked to a hydrophobie group having at least 20 carbon atoms:

   b) a reversibly masked and targeted amphipathîc membrane active polyamine comprising a terpolymer synthesized from primary amine-containing monomers, lower hydrophobie group-containing monomers, and higher hydrophobie group-containing monomers to which a plurality of galactose dérivatives having affinity for the asialoglycoprotein receptor equal to or greater than that of galactose and PEG groups are indivîdually linked to said amphipathîc membrane active polyamine via pH labile dîsubstituted maleamic bonds, respectively, and wherein cleavage of said pH labile dîsubstituted maleamic bonds yields amine groups thereby generating a membrane active polyamine; and,

   c) wherein said oligonucleotide îs not conjugated to said amphipathîc membrane active polyamine.
2. 2. A composition for delivering an oligonucleotide to a liver cell in vivo comprising:

   a) an oligonucleotide covalently linked to a galactose trimer having affinity for the asialoglycoprotein receptor equal to or greater than that of galactose; and,

   b) a reversibly masked and targeted amphipathîc membrane active polyamine comprising a terpolymer synthesized from primary amine-containing monomers, lower hydrophobie group-containing monomers, and higher hydrophobie group-containing monomers to which a plurality of galactose dérivatives and PEG groups are indivîdually linked to said amphipathîc membrane active polyamine via pH labile dîsubstituted maleamic bonds, respectively, and wherein cleavage of said pH labile dîsubstituted maleamic bonds yields amine groups thereby generating a membrane active polyamine; and,

   c) wherein said oligonucleotide is not conjugated to said amphipathîc membrane active polyamine.
3. 3. A composition for delivering an oligonucleotide to a hver cell in vivo comprising:

   _Z(L²-M¹)_y

   N-A + P

   XtL’-M¹),, wherein,

   P is an amphipathîc membrane active polyamine

   L¹ is a pH labile maleamate linkage,

   M¹ is a Charge neutral masking agent containing a galactose dérivative having affinity for the asialoglycoprotein receptor equal to or greater than that of galactose,

   M² is a charge neutral masking agent containing a polyethylene glycol, y and z are integers greater than or equal to zéro wherein the value of y and z together is greater than 50% on amines of P,

   N is an oligonucleotide,

   A is a galactose trimer having affinity for the asialoglycoprotein receptor equal to or greater than that of galactose or a hydrophobie group having at least 20 carbon atoms, positive charge of P is neutralîzed and membrane activity of P is reversîbly inhibited by modification of greater than 50% of the number of primary amines of amphipathic membrane active polyamine P by attachment of M¹ and M²through the maleamate linkages lA and cleavage of l? in response to a decrease in pH restores amines and membrane activity of P.
4. 4. A method of manufacturing an oligonucleotide delivery composition comprising:

   a) forming an ampîhipathic membrane active polyamine;

   b) forming a first masking agent comprising a charge neutral disubstituted maleîc anhydride containing a galactose dérivative having affinity for the asialoglycoprotein receptor equal to or greater than that of galactose;

   c) forming a second masking agent comprising a charge neutral disubstituted maleic anhydride containing a polyethylene glycol;

   d) reversibly inhibiting membrane activity of the amphipathic membrane active polyamine wherein the inhibiting consists of modifying 50% or more of the number of amines on the amphipathic membrane active polyamine by reacting the amphipathic membrane active polyamine with the first and second masking agents thereby linking a plurality of galactose dérivatives and a plurality of polyethylene glycols to the amphipathic membrane active polyamine via physiologieslly pH-labile disubstituted maleamate linkages; and,

   e) linking the oligonucleotide to a galactose trimer having affinity for the asialoglycoprotein receptor equal to or greater than that of galactose or a hydrophobie group having at least 20 carbon atoms;

   f) providing the oligonucleotide and the reversibly inhibited amphipathic membrane active polyamine in solution suitable for administration in vivo.
5. 5. The composition of any of daims 1-4 wherein the oligonucleotide is selected from the group consisting of: DNA, RNA, dsRNA, RNA interférence polynucleotide, siRNA. and miRNA.
6. 6. The composition of any of daims 1 -3 wherein the liver cell consists of a hépatocyte.
7. 7. The composition of any of daims 3 or 4 wherein the amphipathic membrane active polyamine

   5 contains primary amine-containing monomers and hydrophobie group-containing monomers.
8. 8. The composition of daim 7 wherein the amphipathic membrane active polyamine is composed of primary amine-containing monomers, lower hydrophobie group-containing monomers, and higher hydrophobie group-containing monomers.
9. 9. The composition of any of daims 1-4 wherein the reversibly masked amphipathic membrane active polyamine Is soluble in water.

   '
10. 10. The composition of any of daims 1-4 wherein the amphipathic membrane active polyamine is a 15 random copolymer.
11. 11. The composition of claim 10 wherein the random copolymer is selected from the group consisting of polyfvinyl ether) and poly(acrylate).

    20
12. 12. The composition of any of daims 1, 2, or 8 wherein the primary amine-containing monomers, lower hydrophobie group-containing monomers, and higher hydrophobie group-containing monomers are présent in a ratio of 4-8 primary amine-containing monomers : 3-5 lower hydrophobie group-containing monomers : 1 higher hydrophobie group-containing monomers.

    25
13. 13. The composition of daim 12 wherein the lower hydrophobie group consists of a butyl group and the higher hydrophobie group consists of an octadecyl or dodecyl group.
14. 14. The composition of any of daims 3-4 wherein the masking agents are reversibly linked to at least 70% of the number of primary amines on the amphipathic membrane active polyamine.
15. 15. The composition of daim 14 wherein the masking agents are reversibly linked to at least 80% of the number of primary amines on the amphipathic membrane active polyamine.
16. 16. The composition of any of daims 1-15 wherein the reversibly modified amphipathic membrane

    35 active polyamine has a zêta potential between +30 and -30 mV at pH 8.
17. 17. The composition of claim 16 wherein the reversibly modified amphipathic membrane active polyamine has a zêta potential between +20 and -20 mV at pH 8.

    S
18. 18. The composition of claim 17 wherein the reversibly modified amphipathic membrane active polyamine has a zêta potential between +10 and -10 mV at pH 8.
19. 19. The composition of any of daims 1-3 and 5-15 wherein the reversibly modified amphipathic membrane active polyamine has a zêta potential between 0 and -30 mV at pH 8.
20. 20. The composition of claim 19 wherein the reversibly modified amphipathic membrane active polyamine has a zêta potential between 0 and -20 mV at pH 8.
21. 21. The composition of claim 20 wherein thé reversibly modified amphipathic membrane active 15 polyamine has a zêta potential between 0 and -10 mV at pH 8.
22. 22. The composition of any of daims 1-21 wherein the composition Is provided In a pharmaceutically acceptable carrier or diluent.

    20
23. 23. The composition of daim 3 wherein maleamate linkage is a disubstituted maleamate linkage.
24. 24. The composition of claim 3 wherein N is linked to A via a physiologically labile linkage L¹.
25. 25. The composition of claim 24 wherein L¹ is a physiologically labile covalent linkage that is 25 orthogonal to maleamate.
26. 26. The composition of daim 3 wherein A contaîns the hydrophobie group having 20 or more carbon atoms.

    30
27. 27. The composition of daim 3 wherein A contains the galactose trimer having affinity for the asialoglycoprotein receptor equal to or greater than that of galactose.
28. 28. The composition of any of daims 1-3 wherein the ratio of galactose dérivative to PEG linked to the amphipathic membrane active polyamine is 1 to 0.5-2.
29. 29. The composition of any of daims 1-3 and 5-28 wherein the galactose dérivative consists of an N-acetylgalactosamine.
30. 30. The composition of any of daims 2-25 or 27-29 wherein the galactose trimer consists of an

    5 N-acétylgalactosamine trimer.