WO2011140644A1

WO2011140644A1 - Polyacetal or polyketal and ether polymers

Info

Publication number: WO2011140644A1
Application number: PCT/CA2011/000552
Authority: WO
Inventors: Jayachandran Kizhakkedathu; Rajesh Shenoi; Muthiah Manoharan; Rajeev Kallanthottathil; Jayaprakash K. Narayanannair
Original assignee: The University Of British Columbia; Alnylam Pharmaceuticals, Inc.
Priority date: 2010-05-11
Filing date: 2011-05-11
Publication date: 2011-11-17
Also published as: WO2011140644A8

Abstract

The present invention relates to polymers represented by the following formula (I): The invention further provides methods for their preparation and methods for site-specific delivery of nucleic acids by combining them with targeting ligands, endosomolytic ligands and/or PK modulator ligands.

Description

POLYACETAL OR POLYKETAL AND ETHER POLYMERS TECHNICAL FIELD

[0001] The present invention relates to polyacetal or polyketal polymers. The polymers may be useful for delivery of therapeutic agents or nucleic acids.

BACKGROUND

[0002] RNA interference or "RNAi" is a term initially coined by Fire and co-workers to describe the observation that certain double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al. (1998) Nature 391, 806-811). Short double-stranded interfering RNA (dsiRNA) directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi may involve mRNA degradation.

[0003] Work in this field is typified by comparatively cumbersome approaches to delivery of dsRNA to live mammals. E.g., McCaffrey et al. (Nature 418:38-39, 2002) demonstrated the use of dsRNA to inhibit the expression of a luciferase reporter gene in mice. The dsRNAs were administered by the method of hydrodynamic tail vein injections (in addition, inhibition appeared to depend on the injection of greater than 2 mg/kg dsiRNA). The inventors have discovered, inter alia, that the unwieldy methods typical of some reported work are not needed to provide effective amounts of dsiRNA to mammals and in particular not needed to provide therapeutic amounts of dsiRNA to human subjects. The advantages of the current invention include practical, uncomplicated methods of administration and therapeutic applications.

SUMMARY OF THE INVENTION

[0004] The present invention relates to polyacetal or polyketal and ether polymers.

[0005] Compositions comprising polyacetal or polyketal and ether polymers may be combined with one or more therapeutic agents. Methods of using such polymers for delivery of therapeutic agents are also provided. In some embodiments, the therapeutic agents may include nucleic acids. Examples of nucleic acids include iRNAs, siRNAs, single-stranded iRNAs, antagomirs, aptamers, antisense nucleic acids, decoy oligonucleotides, microRNAs (miRNAs), miRNA mimics, antimir, activating RNAs (RNAa), ribozymes, supermirs, Ul adaptor and the like. Derivatives of these nucleic acids may also be used.

[0006] Accordingly, in an embodiment, the invention features a polymer according to formula (I)··

-Y

(I)

wherein

'A' is a polyacetal or polyketal and ether polymer backbone comprising subunits selected from

and combinations thereof; wherein the polymer comprises at least one of

where Ri and R₂ are each independently hydrogen, alkyl, substituted alkyl, or Ri and R₂ can be taken together with the atom they attached to form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl;

X is absent or is selected from C, O, S, alkyl, alkenyl, alkynyl, aryl, substituted alkyl, substituted alkenyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl, heterocycles with at least one heteroatom in the ring, N(R), S-S, C(0)0, C(0)N(R), N(R)C(0)0, N(R)C(0)N(R), OC(O), OC(0)N(R), N(R)C(0), N(R)C(0)N(R), N(R)C(S)N(R), OC(S)N(R), N(R)C(0) or N(R)C(S)0; r, s, and t are each independently 0-10,000; pi to p7 are each independently 0-10;

Y and Z are each independently H, OH or

, where v is 1-10; R]₀ and R₂₀ are each independently H, or formula II;

R is selected from hydrogen, N₃, -NH₂, -NH, COOH, NHS ester, alkane, alkene, alkyne, aldehyde, maleimide, SH, polyethylene glycol (PEG) of varying length and/or MW and -J-W; where J is absent or a linker and W is a therapeutic agent, a targeting ligand, an endosomolytic ligand, a PK (pharmacokinetic) modulator, Z, Y or a combination thereof.

[0007] In another aspect, there is provided a polymer according to formula (II):

(Π)

wherein

X is absent or is selected from C, O, S, alkyl, alkenyl, alkynyl, aryl, substituted alkyl, substituted alkenyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,

heterocycloalkyl, or substituted heterocycloalkyl, heterocycles with at least one heteroatom in the ring, N(R), S-S, C(0)0, C(0)N(R), N(R)C(0)0, N(R)C(0)N(R), OC(O), OC(0)N(R), N(R)C(0), N(R)C(0)N(R), N(R)C(S)N(R), OC(S)N(R), N(R)C(0) or N(R)C(S)0;

Ri and R₂ are each independently hydrogen, alkyl, substituted alkyl, or R_\ and R₂ can be taken together with the atom they attached to form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl; r, s, and t are each independently 0-10,000; q is 5-30,000; pi to p7 are each independently 0-10;

Y and Z are each independently H, OH or

where v is 1-10; Ri₀ and R₂₀ are each independently H, or formula II;

Y and Z are each independently H, OH or

where v is 1-10; R₁₀ and

R₂₀ are each independently H, or formula II;

R is selected from hydrogen, N₃, -N¾, -NH, COOH, NHS ester, alkene, alkyne, aldehyde, maleimide, SH, PEG of varying length and/or MW and -J-W; where J is absent or a linker and W is a therapeutic agent, a targeting ligand, an endosomolytic ligand, a PK (pharmacokinetic) modulator, Z, Y or a combination thereof.

[0008] In another aspect, there is provided a polymer according to formula (III):

(III)

wherein

heterocycloalkyl, or substituted heterocycloalkyl, heterocycles with at least one heteroatom in the ring, N(R), S-S, C(0)0, C(0)N(R), N(R)C(0)0, N(R)C(0)N(R), OC(O), OC(0)N(R), N(R)C(0), N(R)C(0)N(R), N(R)C(S)N(R), OC(S)N(R), N(R)C(0) or N(R)C(S)0;. Ri and R₂ are each independently alkyl, substituted alkyl, or Rj and R₂ can be taken together with the atom they attached to form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl; r, s, and t are each independently 0-10,000; q is 5-30,000; pi to p7 are each independently 0-10;

Y and Z are each independently H, OH or

, where v is 1-10; R₁₀ and

R₂o are each independently H, or formula II;

[0009] R is selected from hydrogen, N₃, -NH₂, -NH, COOH, NHS ester, alkene, alkyne, aldehyde, maleimide, SH, PEG of varying length and/or MW and -J-W; where J is absent or a linker and W is a therapeutic agent, a targeting ligand, an endosomolytic ligand, a PK (pharmacokinetic) modulator, Z, Y or a combination thereof.

[0010] In another aspect, there is provided a polymer according to formula (IV):

(IV)

wherein

Ri and R₂ are each independently alkyl, substituted alkyl, or R_\ and R₂ can be taken together with the atom they attached to form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl; q is 5-30,000; pi to p4 are each independently 0-10; nl to n2 each independently 0-10,000;

Y and Z are each independently H, OH or

where v is 1 -10;

Rio and R₂₀ are each independently H, or formula II; R is selected from hydrogen, N₃, -NH₂, -NH, COOH, NHS ester, alkene, alkyne, aldehyde, maleimide, SH, PEG of varying length and/or MW and -J-W; where J is absent or a linker and W is a therapeutic agent, a targeting ligand, a masking agent, an endosomolytic ligand, a PK (pharmacokinetic) modulator, Z, Y or a combination thereof. [001 1 ] In another aspect, there is provided a polymer according to formula (V):

(V)

wherein X is absent or is selected from C, O, S, alkyl, alkenyl, alkynyl, aryl, substituted alkyl, substituted alkenyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,

Ri and R₂ are each independently hydrogen, alkyl, substituted alkyl, or Ri and R₂ can be taken together with the atom they attached to form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl; r, s, and t are each independently 1-10,000; q is 5-30,000; v is 1-10; pi to p21 are each independently 0-10; ml, m2 or m3 each independently 1-20;

R is selected from hydrogen, N₃, -NH₂, -NH, COOH, NHS ester, alkene, alkyne, aldehyde, maleimide, SH, PEG of varying length and/or MW and -J-W; where J is absent or a linker and W is a therapeutic agent, a targeting ligand, an endosomolytic ligand, a PK (pharmacokinetic) modulator, Z, Y or a combination thereof;

Yl, Y2 and Y3 are each independently H, OH or

, where v is 1-10; and

Rio and R₂₀ are each independently H, or formula II;

[0012] In another aspect, there is provided a composition comprising a polymer according to one or more of Formula I, II, III, IV or V in combination with a nucleic acid. [0013] In some embodiments of the invention, the polymer may be a homopolymer, random copolymer, tercopolymer, block copolymer, linear, branched, dendritic, amphiphilic, matrix, block or crossed-linked polymer.

[0014] In some embodiments of the invention, the endosomolytic ligand may be selected from the group consisting of imidazoles, poly or oligoimidazoles, linear or brached polyethyleneimines (PEIs), linear and branched polyamines, cationic linear and branched polyamines,

polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals, polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, and natural and synthetic fusogenic lipids.

[0015] In some embodiments of the invention, the endosomolytic ligand may be a polyanionic peptide or a polyanionic peptidomimetic.

[0016] In some embodiments of the invention, the endosomolytic ligand may be selected from the group consisting of GALA, EALA, INF-7, Inf HA-2, diINF-7, diINF3, GLF, GALA-INF3, INF-5, JTS-1, ppTGl, ppTG20, KALA, HA, melittin, and histidine-rich peptide CH ^HC.

[0017] In some embodiments of the invention, the targeting ligand may be selected from the group consisting of an antibody, a ligand-binding portion of a receptor, a ligand for a receptor, an aptamer, D-galactose, N-acetyl-D-galactose (GalNAc), multivalent N-acetyl-D-galactose, D- mannose, cholesterol, a fatty acid, a lipoprotein, folate, thyrotropin, melanotropin, surfactant protein A, mucin, carbohydrate, multivalent lactose, multivalent galactose, N-acetyl- galactosamine, N-acetyl-gulucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipophilic moiety that enhance plasma protein binding, a steroid, bile acid, vitamin Bj₂, biotin, an RGD peptide, an RGD peptide mimetic, ibuprofen, naproxen, aspirin, folate, and analogs and derivatives thereof.

[0018] In some embodiments of the invention, the targeting ligand may be selected from the group consisting of D-galactose, N-acetyl-D-galactose (GalNAc), multivalent N-acetyl-D- galactose, cholesterol, folate, and analogs and derivates thereof. [0019] In some embodiments of the invention, the nucleic acid may be selected from a group consisting of an iRNA agent, an antisense oligonucleotide, an antagomir, an activating RNA, a decoy oligonucleotide, an aptamer, and a ribozyme.

[0020] In some embodiments of the invention, the nucleic acid down may regulate the expression of a target gene through an RNA interference mechanism.

[0021] In some embodiments of the invention, the nucleic acid may be a single-stranded oligonucleotide.

[0022] In some embodiments of the invention, the nucleic acid may be a double-stranded oligonucleotide. [0023] In some embodiments of the invention, the linker may be a redox cleavable linker.

[0024] In some embodiments of the invention, the linker may comprise a pH sensitive component.

[0025] In some embodiments, the targeting ligand provides sufficient permeability and retention to allow the nucleic acid to accumulate in the cell. [0026] In another aspect of the invention, there is provided a method of inhibiting the expression of one or more genes, the method comprising contacting one or more cells with a polymer according to one or more of Formula I, II, III, IV or V, in combination with a effective amount of nucleic acid, wherein the effective amount is an amount that suppresses the expression of the one or more genes. [0027] In some embodiments of the invention, the method may further comprise allowing the cell to internalize the polymer.

[0028] The details of one or more embodiments of the invention are set forth in the

accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from this description, and from the claims. A person of ordinary skill in the art will readily recognize that additional embodiments of the invention exist. This application incorporates all cited references, patents, and patent applications by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0030] Figure 1 shows an exemplary synthetic scheme for the ketal monomer 5 (monoepoxide). [0031] Figure 2 shows an Ή NMR spectrum of crude monomer (in CDC1₃).

[0032] Figure 3 shows an Ή NMR spectrum of monoepoxide (ketal monomer) in CDC1₃.

[0033] Figure 4 shows an 1H NMR spectrum of diepoxide (in CDCI3).

[0034] Figure 5 shows an Ή NMR spectrum of a PKHA homopolymer (in CDC1₃).

[0035] Figure 6 shows a synthetic scheme of poly (ketal hydroxyl alkane) (PKHA)

homopolymer.

[0036] Figure 7 shows GPC traces of a 29000 Da homopolymer assayed in a) 0.1 M NaN0₃ (no buffering) or b) 0.1 M NaN03, buffered to pH 8.5 with phosphate buffer.

[0037] Figure 8 shows a synthetic scheme of a PKHA copolymer.

[0038] Figure 9 shows an Ή NMR spectrum of a PKHA copolymer in CDC1₃. [0039] Figure 10 shows a gel permeation chromatography (GPC) trace of a PKHA copolymer in 0.1 M NaN0₃ at pH 8.5.

[0040] Figure 11 shows Ή NMR (D₂0, 300 MHz): δ ppm 1.37 (ketal CH₃ protons), 3.2-4.0 (CH₂ and CH protons from glycidol and ketal monomer).

[0041] Figure 12 shows a GPC trace of PKHA copolymer (in 0.1 M NaN0₃ at pH 8.5). [0042] Figure 13 shows an Ή NMR spectrum of a 480 kDa PKHA copolymer in D₂0

[0043] Figure 14 shows a plot of the kinetics of degradation of poly(ketal hydroxyalkanes) polymer as measured from 1H NMR peak integrations at different pHs and time points. The polymer degraded only 8% in D₂0 after 72 h. [0044] Figure 15 an Ή NMR spectrum of poly(ketal hydroxyalkanes) polymer degradation with time at pH 5.5.

[0045] Figure 16 shows an Ή NMR spectrum of poly (ketal hydroxyalkanes) polymer in D₂0.

[0046] Figure 17 shows a pH dependent degradation profile of 45 KDa PKHA homopolymer. [0047] Figure 18 shows a comparison of degradation of 13 KDa and 45KDa PKHA

homopolymers at pH 6.4.

[0048] Figure 19 shows a pH dependent degradation profile for a 22 KDa PKHA copolymer.

[0049] Figure 20 shows a comparison of degradation of 8.5 KDa and 22 KDa PKHA copolymers at pH 6.0. [0050] Figure 21 shows a scheme of a chemical modification of PKHA homo and copolymers.

[0051] Figure 22 shows an Ή NMR spectrum of phthalimide functionalized PKHA polymer.

[0052] Figure 23 shows an Ή NMR spectrum of an azide functionalized PKHA polymer.

[0053] Figure 24 shows an Ή NMR spectrum of PKHA polymer (Example 13).

[0054] Figure 25 shows an Ή NMR spectrum of PKHA polymer (Example 18). [0055] Figure 26 shows an Ή NMR spectrum of PKHA polymer (Example 21).

[0056] Figure 27 shows an ^[H NMR spectrum of PKHA polymer (Example 22).

[0057] Figure 28 shows an Ή NMR spectrum of PKHA polymer (Example 24).

[0058] Figure 29 shows a GPC-MALLS trace of PKHA polymer (Example 18) in chloroform.

[0059] Figure 30 shows a GPC-MALLS trace of PKHA polymer (Example 21) in chloroform. [0060] Figure 31 shows a GPC-MALLS trace of PKHA polymer (Example 22) in chloroform.

[0061] Figure 32 shows pH dependent degradation of PKHA polymer at 25°C (Example 13).

[0062] Figure 33 shows pH dependent degradation of PKHA polymer at 25°C (Example 18). [0063] Figure 34 shows pH dependent degradation of PKHA polymer at 25°C (Example 21).

[0064] Figure 35 shows comparison of degradation of different PKHA polymers at pH 5.5 and 25°C.

[0065] Figure 36 shows comparison of degradation of different PKHA polymers at pH 6.5 and 25°C.

[0066] Figure 37 shows pH dependent degradation of PKHA polymer (Example 13) at 37°C.

[0067] Figure 38 shows pH dependent degradation of PKHA polymer (Example 18) at 37°C.

[0068] Figure 39 shows degradation of PKHA polymer (Example 21) at pH 4.1 and 37°C.

[0069] Figure 40 shows comparison of degradation of different PKHA polymers at pH 1.1 and 37°C.

[0070] Figure 41 shows degradation of PKHA polymer (Example 22) at pH 5.5 and 37°C. [0071] Figure 42 shows a general scheme for conjugation of siRNA to polyketal polymers. [0072] Figure 43 shows in vitro data for Dimethyl ketal polymer siRNA conjugates. [0073] Figure 44 shows in vitro data for Dimethyl ketal polymer siRNA conjugates. [0074] Figure 45 shows a GPC trace of polymer conjugate as described in Example 28. DETAILED DESCRIPTION

[0075] The present invention relates to polyacetal, polyketal and ether polymers. In particular polymers according to any of Formulae I, II, III, IV and/or V.

[0076] In the description that follows, a number of terms are used to facilitate understanding of various aspects of the invention. Use of examples in the specification, including examples of terms, is for illustrative purposes only and is not intended to limit the scope and meaning of the embodiments of the invention herein.

[0077] Polymers as described herein may have a molecular weight, or an average molecular weight, of about 5k to about 1500 k, or any amount therebetween, or from 10k to about 1000 k or any amount therebetween; from about 20 k to about 750 k or any amount therebetween; from about 40 k to about 500 k, or any amount therebetween; or from about 35 k to about 90 k, or any amount therebetween. For example, the average molecular weight of the polymers may be 5,10,15, 20, 25,30, 35, 40, 45, 50,60, 65, 70, 76, 80, 85, 90, 95, 100, 125, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 kDa, or any amount therebetween.

[0078] Polymers as describe herein may have a degree of polymerization, or an average degree of polymerization, of about 20 to about 500, or any amount therebetween, or from about 75 to about 400, or from about 100 to about 300, or from about 150 to about 200, or any amount

therebetween. For example, the average degree of polymerization of the polymers may be 25, 40, 50, 150, 170, 180, 190, 200, 205, 210, 220, 230, 240, 250, 275, 300, or any amount

therebetween.

[0079] Polymers as described herein may have a polydispersity index of about 1 to about 10, or any amount therebetween. For example, the polydispersity index may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any amount therebetween. In some embodiments, the polydispersity index may be about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or any amount therebetween.

[0080] The molecular weight of a polymer may depend on the amount of monomer, the rate at which it is added, or a combination thereof, for a given amount of initiator. For example, adding monomer to the reaction at a faster rate, or as a single bolus, may increase the polydispersity of the polymer.

[0081] Polymers as described herein may be soluble in a variety of organic solvents (for example, chloroform, dichloromethane, methanol, Ν,Ν-dimethyl formamide, tetrahydrofuran or the like) and in aqueous solution (for example water, buffer, saline or the like).

[0082] Another exemplary embodiment of the invention provides for a method of synthesizing poly(ketal hydroxyalkane (PKHA) homopolymer by polymerization of 2- { 1 -methyl- 1 -[2-(oxiran- 2-yl methoxy) ethoxy] ethoxy} ethanol (compound 5). A mixture of 1,1,1 - Tris(hydroxymethyl)propane and initiator potassium methylate in methanol is stirred under argon, followed by removal of methanol by vacuum. The mixture is heated to a temperature of about 70 to about 120°C, and compound 5 is added over 3-24 hours, The resulting polymer is resolubilized, precipitated and dried.

[0083] The resulting polymers may have a Mn of about 6000 Da to about 45000 Da, with a polydispersity of about 1.22 to about 1.7, or any amounts therebetween. [0084] Another embodiment of the invention provides for a method of synthesizing poly(ketal hydroxyalkane (PKH A) copolymer by polymerization of 2- { 1 -methyl- 1 -[2-(oxiran-2-yl methoxy) ethoxy] ethoxy} ethanol (compound 5) and glycidol. A mixture of 1,1 ,1 - Tris(hydroxymethyl)propane and initiator potassium methylate in methanol is stirred under argon, followed by removal of methanol by vacuum. The mixture is heated to a temperature of about 70 to about 120°C, and compound 5 and glycidol in a molar ration of about 3:1 to about 1 :3, to about 1 :9 is added over 3-24 hours. The resulting polymer is resolubilized, precipitated and dried.

[0085] The resulting polymers may have a Mn of about 6000 Da to about 480000 Da, with a polydispersity of about 1.1 to about 2.0, or any amounts therebetween. [0086] In some embodiments, at least 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the hydroxyl groups (-X-R; where 'R'= H in formula II- V) of the polymer are modified to amines, alkyl or aryl substituted amines, carboxylic acids, azides, aldehydes, maleimides, thiols, NHS ester, aromatics, alkenes and alkynes.

[0087] In some embodiments, the polymers of the invention may be processed to form particles, sutures, bulk materials, tissue engineering scaffolds, or implants. Processing may include incorporating the polymers into an implantable device, encapsulating particular polymers in carriers such as liposomes, membranes or the like, or otherwise manipulating the polymers to facilitate storage or administration to a subject.

[0088] In one exemplary embodiment, one or more of the polymers further comprise a bioactive material conjugated or entrapped therein.

[0089] In one exemplary embodiment of the invention, there is provided a composition comprising a polymer according to Formula I, II, III, IV or V, and a nucleic acid. [0090] A "bioactive material" may be one or more than one of a polynucleotides, a nucleic acid, an oligonucleotide, a polypeptide, a protein, a peptide, an antibody, a vaccine, an antigen, a genetic agent, a small molecule drug, or a therapeutic agent.

[0091] In an exemplary embodiment of the invention, a therapeutic agent may be covalently linked (or conjugated) to the polymer, or covalently linked to a linker, the linker in turn covalently linked to the polymer. As set forth herein, the linker may be cleavable.

[0092] In another exemplary embodiment of the invention, a therapeutic agent may be non- covalently linked to the polymer, or non-covalently linked to a linker, the linker in turn covalently linked to the polymer. Again, the linker may be cleavable. The non-covalent linkage (non-covalent interaction or non-covalent association) may be include electrostatic, induction or dispersion or Van Der Waals interaction between the therapeutic agent and the polymer, and may include, or leasd to hydrogen bonding, charge transfer and/or hydrophobic effects between the therapeutic agent and the polymer. The stability of such non-covalent interactions may vary with the surrounding environment e.g. solvent, temperature, pH, other molecules in the solvent, or the like. The stability of complexes formed by such non-covalent interactions may be assessed by any of several methods known in the art; selection of a suitable method will be within the ability of one skilled in the art, and may depend on the nature of the complex and surrounding involvement. Methods include, for example, spectrometry, potentiometry, solubility, chemical reactivity or the like. [0093] For an exemplary embodiment where the therapeutic agent is a nucleic acid, and the nucleic acid forms a non-covalent complex with an acid-labile polymer according to one of the exemplary embodiments described herein, the polymer-nucleic acid complex may be

phagocytosed by a cell. Within the endosome, the polymer-nucleic acid complex is exposed to a lower pH and the acid-labile polymer degrades. As the polymer degrades, the nucleic acid is released into the endosome and from there, into the intracellular environment. If the nucleic acid is an siRNA, it may interact with an RNA transcript comprising a sequence that hybridizes with the siRNA, and interact with a RISC complex within the cell and target the RNA transcript for degradation. Degradation of the RNA transcript may selectively and specifically reduce the amount of RNA transcript (e.g. mRNA) available for translation into a corresponding

polypeptide, thereby decreasing the amount of the polypeptide available to the cell. [0094] For an exemplary embodiment where the therapeutic agent is covalently linked to the acid-labile polymer, a similar series of events may occur. The polymer-therapeutic agent may be phagocytosed by a cell, and within the endosome, the polymer-nucleic acid complex is exposed to a lower pH and the polymer degraded. As the polymer degrades, the therapeutic agent is released into the endosome and from there, into the intracellular environment. If the therapeutic agent is an siRNA, interaction with the RISC complex may occur; if the therapeutic agent is a small molecule or drug or other agent, it may interact with one or more polypeptides, enzymes or the like in the cell and exert a biological effect in that manner. In some embodiments, the therapeutic agent may retain a small portion of the polymer or linker to which it was attached e.g. one or a few carbon moieties or the like. The 'small portion' may have no impact on the biological effect of the therapeutic agent.

[0095] The terms "nucleotide polymer", "oligonucleotide", "oligonucleotide polymer",

"oligonucleotide", "nucleic acid", "oligomer" or "nucleic acid polymer" are used

interchangeably, and refer to polymers comprising at least two nucleotides. A nucleic acid may comprise a single species of DNA monomer, RNA monomer, or may comprise two or more species of DNA monomer, or RNA monomers in any combination, including DNA or RNA monomers with modified intemucleoside linkages or 'backbones'. Nucleic acid may be single or double-stranded, for example, a double-stranded nucleic acid molecule may comprise two single- stranded nucleic acids that hybridize through base pairing of complementary bases. The nucleic acid may comprise a sequence that hybridizes with a specific sequence of a target nucleic acid (e.g. an antisense sequence or an siRNA) to alter the transcription or translation of the target nucleic acid. The nucleic acid may comprise one or more coding sequences for a polypeptide, enzyme, protein, receptor, hormone or the like. The nucleic acid may comprise a sequence for other motifs or nucleic acid structures of interest including, for example transcription or translational regulatory elements (for example, a promoter, an enhancer, a terminator, one or more signal sequences or the like), iRNA, vectors, plasmids, siRNA, microRNAs (miRNAs), short inactivating nucleic acid (siNA), nucleic acid stem-loop structures, short hairpin RNAs (shRNAs), aptamers, supermirs, ribozymes, antagomirs, adapters, activating RNA, decoy oligonucleotide, iRNA agent or the like. [0096] The term "subject" or "patient" generally refers to mammals and other animals including humans and other primates such as chimpanzees or monkeys, companion animals, zoo, and farm animals, including, but not limited to, cats, dogs, rodents, rats, mice, hamsters, rabbits, horses, cows, sheep, pigs, goats, poultry, etc. A subject includes one who is to be tested, or has been tested for prediction, assessment or diagnosis of a disease or disorder, or for response to a therapeutic agent. The subject may have been previously assessed or diagnosed using other methods, such as those described herein or those in current clinical practice, or may be selected as part of a general population (a control subject).

[0097] In one embodiment, the polymer of the invention comprises pH-sensitive functional groups, whereby the functional groups are designed to remain relatively stable in plasma at neutral physiological pH (about 7.4), but degrade by hydrolysis in the more acidic environment of about pH 5.0-6.5, thereby resulting in degradation upon hydrolysis. In one instance, the polymer is processed to deliver a bioactive material, whereupon the bioactive material is released in response to mildly acidic conditions, found in the body such as in tumors, inflammatory tissues and in cellular compartments such as lysosomes and phagolysosomes of antigen presenting cells.

[0098] In one embodiment, the particle size formed from the polymer of the invention is about 3 nm to about 50 μηι, or about 3 nm to about 2000 nm, or about 40 to about 300 nm. A variety of suitable methods are known in the art for assessment of particle size, for example,

photoanalytical methods, laser diffraction methods, or the like.

[0099] In one embodiment, a linker includes, but not limited to, an organic moiety that connects two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR¹, S-S, C(O), C(0)N(R'), N(R^L)C(0), SO, S0₂, S0₂NH, -OP(0)(OR^Q)- 0-, -OP(0)(SR^q)0-, -OP(S)(SR^q)0-, -SP(0)(0R^Q)O, -SP(0)(OR^Q)S-, -OP(0)(NR')0-, -N(R')- P(0)(OR^q)0-, -OP(0)(OR^Q)N(R')-, -C(R^P ₂)P(0)(OR^q)-0-, -C(R^P ₂)P(0)(SR^q)-0-, - C(R^P ₂)P(0)(NR')-0-, -OP(0)(OR^Q )C(R^P ₂)-, -OP(0)(SR^Q)C(R^P ₂)-, OP(0)(NR¹)C(R^P ₂)-, - OC(0)N(R')-, -0C(NR')N(R¹)-, -OC(S)N(R')-, -N(R')C(0)0-, -N(R¹)C(S)0-, - N(R')C(0)N(R')- ,or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylaryl alkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,

alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl,

alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl,

alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,

alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,

> alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), S0₂, NiR^, C(0), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle;

[00100] where R¹ is hydrogen, acyl, aliphatic or substituted aliphatic. It is further

) understood that the linker can be non-cleavable or can be cleavable.

[00101] In one embodiment, the linker is Ci-C3₀ alkyl, optionally interrupted with at least one O, S, NR, N(CO)0, N(CO), PS₂, phosphate, phosphorothioate, phosphonate or combinations thereof. It is understood that any moieties described herein can be connect via both directions, i.e. N(CO)0 can be N(CO)0 or 0(CO)N.

5 [00102] In one embodiment, the linker is represented by structure

-[V-Q,-R]_q-T-,

wherein:

V, R and T are each independently for each occurrence absent, CO, NH, O, S, S-S, OC(O), NHC(O), CH₂, CH₂NH, CH₂0; N ^a)C(0), -C(0)-CH(R^a)-NH-, -C(0)-(optionally

⁾ , acetal, ketal,

Qi is independently for each occurrence absent, -(CH₂)_n-, -C(R¹⁰⁰)(R²⁰⁰)(CH₂)_n-, - (CH₂)_nC(R¹⁰⁰)(R²⁰⁰)-, -(CH₂CH₂0)_mCH₂CH₂-, or -(CH₂CH₂0)_mCH₂CH₂NH-;

R^a is H or an amino acid side chain; R'⁰⁰ and R²⁰⁰ are each independently for each occurrence H, CH₃, OH, SH or N(R^X)₂;

R^X is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl or benzyl; q is independently for each occurrence 0-20; n is independently for each occurrence 1 -20; and m is independently for each occurrence 0-50.

[00103] In one embodiment, the linker has the structure -[(P-Q_rR)_q-X-(P'-Qi '-R')q ]q"-T, wherein:

P, R, T, P', R' and T' are each independently for each occurrence absent, CO, NH, O, S, OC(O),

NHC(O), CH₂, CH₂NH, CH₂0; NHCH(R^a)C(0), -C(0)-CH(R^a)-NH-, -C(0)-(optionally

) substituted alkyl)-NH-, acetal, ketal , CH=N-0 ,

Qi and Qj' are each independently for each occurrence absent, -(CH₂)_n-, -C(R¹⁰⁰)(R²⁰⁰)(CH₂)_n-, - (CH₂)_nC(R¹⁰⁰)(R²⁰⁰)-, -(CH₂CH₂0)mCH₂CH₂-, or -(CH₂CH₂0)_mCH₂CH₂NH-;

X is a cleavable linker; > R^a is H or an amino acid side chain;

R¹⁰⁰ and R²⁰⁰ are each independently for each occurrence H, CH₃, OH, SH or N(R^X)₂;

R is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl or benzyl; q, q' and q' are each independently for each occurrence 0-20; n is independently for each occurrence 1-20; and ⁾ m is independently for each occurrence 0-50. " " is the polymer backbone defined by any one or more of formula II to V.

[00104] In one embodiment, the linker may be cleavable.

[00105] In one example, the polymer of the invention comprises various combinations of the following features located on the surface or interior or distributed throughout. In one embodiment, the R group is selected from the group consisting of:

[00106] Endosomolytic ligands For macromolecular drugs and hydrophilic drug molecules, which cannot easily cross bilayer membranes, entrapment in endosomal/lysosomal compartments of the cell is thought to be the biggest hurdle for effective delivery to their site of action. In recent years, a number of approaches and strategies have been devised to address this problem. For li e endosomolytic activity through protonation and/or pH-induced conformational changes, include charged polymers and peptides. Examples may be found in Hoffman, A. S., Stayton, P. S. et al. (2002). Design of "smart" polymers that can direct intracellular drug delivery. Polymers Adv. Technol. 13, 992-999; Kakudo, Chaki, T., S. et al. (2004). Transferrin-Modified Liposomes Equipped with a pH-Sensitive Fusogenic Peptide: An Artificial Viral-like Delivery System. Biochemistry 436, 5618-5628; Yessine, M. A. and Leroux, J. C. (2004). Membrane-destabilizing polyanions:

interaction with lipid bilayers and endosomal escape of biomacromolecules. Adv. Drug Deliv. Rev. 56, 999-1021 ; Oliveira, S., van Rooy, I. et al. (2007). Fusogenic peptides enhance endosomal escape improving siRNA-induced silencing of oncogenes. Int. J. Pharm. 331, 211-4. They have generally been used in the context of drug delivery systems, such as liposomes or lipoplexes. For folate receptor-mediated delivery using liposomal formulations, for instance, a pH-sensitive fusogenic peptide has been incorporated into the liposomes and shown to enhance the activity through improving the unloading of drug during the uptake process (Turk, M. J., Reddy, J. A. et al. (2002). Characterization of a novel pH-sensitive peptide that enhances drug release from folate-targeted liposomes at endosomal pHs. Biochim. Biophys. Acta 1559, 56-68)

[00107] In certain embodiments, the endosomolytic ligands of the present invention may be polyanionic peptides or peptidomimetics which show pH-dependent membrane activity and/or fusogenic activity. A peptidomimetic may be a small protein-like chain designed to mimic a peptide. A peptidomimetic may arise from modification of an existing peptide in order to alter the molecule's properties, or the synthesis of a peptide-like molecule using unnatural amino acids or their analogs. In certain embodiments, they have improved stability and/or biological activity when compared to a peptide. In certain embodiments, the

endosomolytic ligand assumes its active conformation at endosomal pH (e.g., pH 5-6). The "active" conformation is that conformation in which the endosomolytic ligand promotes lysis of the endosome and/or transport of the modular composition of the invention, or its any of its components (e.g., a nucleic acid), from the endosome to the cytoplasm of the cell. An endosomal release agent is an agent that initiates, promotes, or initiates and promotes lysis of an endosome, and/or release of endosomal contents.

[00108] Libraries of compounds may be screened for their differential membrane activity at endosomal pH versus neutral pH using a hemolysis assay. Promising candidates isolated by this method may be used as components of the modular compositions of the invention. A method for identifying an endosomolytic ligand for use in the compositions and methods of the present invention may comprise: providing a library of compounds; contacting blood cells with the members of the library, wherein the pH of the medium in which the contact occurs is controlled; determining whether the compounds induce differential lysis of blood cells at a low pH (e.g., about pH 5-6) versus neutral pH (e.g., about pH 7-8). [00109] Exemplary endosomolytic ligands include the GALA peptide (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al., J. Am. Chem. Soc, 1996, 118: 1581-1586), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). In certain embodiments, the endosomolytic ligand may contain a chemical group (e.g., an amino acid) which will undergo a change in charge or protonation in response to a change in pH. The endosomolytic ligand may be linear or branched. PCT Publication WO 2009/126933 describes entosomolytic components for site-specific delivery of nucleic acids. Examples of endosomolytic ligands are provided in Table 1. Table 1. Exemplary Endosomolytic ligands

Nle = norleucine

[00110] SEQ ID NOs: 6, 7 and 10 are dimer peptides. diINF-7 is a dimer of INF-7, covalently linked by a C-terminal disulfide bond between the cysteine residues; diLNF3 is a dimer of INF-3, covalently linked by a C-terminal disulfide bond between the cysteine residues. INF-5 is a head-to-head dimer peptide, with a carboyl-terminal lysine as a linking amino acid. Methods of synthesis of these dimer peptides is found, for example, in Plank et al., 1994 (J. Biol Chem 26917):12918-12924).

[001 11] In some embodiments, endosomolytic ligands can include imidazoles, poly or oligoimidazoles, linear or branched polyethyleneimines (PEIs), linear and branched polyamines, e.g. spermine, cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic

peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.

[00112] The endosomolytic ligand of this invention is a cellular compartmental release component, and may be any compound capable of releasing from any of the cellular

compartments known in the art, such as the endosome, lysosome, endoplasmic reticulum (ER), golgi apparatus, microtubule, peroxisome, or other vesicular bodies with the cell. [00113] In some embodiments, the membrane active functionality [e.g. alkyl group, peptides] of the endosomolytic ligand is masked when the endosomolytic ligand is conjugated with an oligonucleotide. When the oligonucleotide reaches the endosome, the membrane active functionality is unmasked and the endosomlytic ligand becomes active. The unmasking may be carried out more readily under the conditions found in the endosome than outside the endosome. For example, the membrane active functionality can be masked with a molecule through a cleavable linker that under goes cleavage in the endosome. Without wishing to be bound by theory, it is envisioned that upon entry into the endosome, such a linkage may be cleaved and the masking agent released from the endosomolytic ligand.

[001 14] In some embodiments, the masking agent may have a cleavable linker that, upon cleavage, release a functional group that can cleave the linkage between the masking agent and the active functional group of the endosomolytic ligand. One example is a masking agent linked to the endosomolytic ligand through a amide type linkage, and having a S-S bond. Upon entry into the endosome, the S-S bond can be cleaved releasing free thiols that can then cleave the amide linkage between the masking agent and the endosomolytic ligands either inter or intra molecularly. United States Patent Application Publication No. 2008/0281041 (which is incorporated herein by reference) describes examples of masked endosomolytic polymers that may be amenable to the present invention.

[00115] Lipids having membrane activity are also amenable to the present invention as endosomolytic ligands. Such lipids may also be described as fusogenic lipids. Fusogenic lipids fuse with and consequently destabilize a membrane. Fusogenic lipids may comprise unsaturated acyl chains. Exemplary fusogenic lipids include l,2-dileoyl-sn-3-phosphoethanolamine

(DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31 -tetraen- 19-ol (Di-Lin), N-methyl(2,2-di((9Z, 12Z)- octadeca-9,12-dienyl)-l,3-dioxolan-4-yl)methanamine (DLin-k-DMA) and N-methyl-2-(2,2- di((9Z, 12Z)-octadeca-9, 12-dienyl)-l ,3-dioxolan-4-yl)ethanamine (XTC).

[00116] The histidine-rich peptide H5WYG (Midoux et al., 1998. Bioconjug Chem

9(2):260-7) is a derivative of the N-terminal sequence of the HA-2 subunit of the influenza virus hemagglutinin in which 5 of the amino acids have been replaced with histidine residues, and is an example of a fusogenic peptide. Protonation of the histidine residues of the H5WYG peptide may selectively destabilize membranes at a slightly acidic pH.

[001 17] In some embodiments, the endosomolytic ligand is a cell-permeation agent, preferably a helical cell-permeation agent. The agent may be an amphipathic peptide. The helical agent may be an alpha-helical peptide, which may comprises a lipophilic phase and a lipophobic phase. A cell-permeation agent can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide or hydrophobic peptide, e.g. comprising of Tyr, Trp and Phe, dendrimer peptide, constrained peptide or crosslinked peptide. In some embodiments, the cell permeation peptide can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence

AAVALLPAVLLALLAP (SEQ ID NO:33). An RFGF analogue {e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 34) containing a hydrophobic MTS can also be a targeting ligand. The cell permeation peptide can be a "delivery" peptide, for conveying large polar molecules including peptides, polypeptides, oligonucleotides, nucleic acids or proteins across cell membranes. Some exemplary cell-permeation peptides are shown in Table 2.

[00118] Table 2. Exemplary Ceil Permeation Peptides.

Peptide name SEQ ID

Amino acid Sequence Reference

NO:

[00119] Cell-permeation peptides can be linear or cyclic, and include D-amino acids, non- peptide or pseudo-peptide linkages, or peptidyl mimics. In addition the peptide and peptide mimics can be modified, e.g. glycosylated or methylated. Synthetic mimics of targeting peptides are also included.

[00120] In certain embodiments, more than one endosomolytic ligand may be incorporated in the modular composition of the invention as described in one or more of formula I to V, or a combination thereof. In some embodiments, this may entail incorporating more than one of the same endosomolytic ligand into the modular composition. In other embodiments, this may entail incorporating two or more different endosomolytic ligands into the modular composition.

[00121 ] These endosomolytic ligands may mediate endosomal escape by, for example, changing conformation at endosomal pH. In certain embodiments, the endosomolytic ligands may exist in a random coil conformation at neutral pH and rearrange to an amphipathic helix at endosomal pH. As a consequence of this conformational transition, these peptides may insert into the lipid membrane of the endosome, causing leakage of the endosomal contents into the cytoplasm. As the conformational transition is pH-dependent, the endosomolytic ligands may display little or no fusogenic activity while circulating in the blood (pH -7.4). Fusogenic activity is defined as that activity which results in disruption of a lipid membrane by an endosomolytic ligand. One example of fusogenic activity is the disruption of the endosomal membrane by the endosomolytic ligand, leading to endosomal lysis or leakage and transport of one or more components of the modular composition of the invention (e.g., the nucleic acid) from the endosome into the cytoplasm.

[00122] In addition to the hemolysis assay described herein, suitable endosomolytic ligands can be tested and identified by a skilled artisan using other methods. For example, the ability of a compound to respond to a change in pH environment (e.g. a change in the charge on the compound) can be tested by routine methods, e.g., in a cellular assay. As an example, a cell may be combined with, or contacted by a test compound, and the cell allowed to internalize the test compound, e.g., by endocytosis. An endosome preparation can then be made from the contacted cells, and the endosome preparation compared to an endosome preparation from control cells. A change, e.g., a decrease, in the endosome fraction from the contacted cell vs. the control cell indicates that the test compound can function as a fusogenic agent. Alternatively, the contacted cell and control cell can be evaluated, e.g., by microscopy, e.g., by light or electron microscopy, to determine a difference in the endosome population in the cells. The test compound and/or the endosomes can labeled, e.g., to quantify endosomal leakage.

[00123] In another type of assay, a modular composition described herein is constructed using one or more test or putative fusogenic agents. The modular composition can be constructed using a labeled nucleic acid. The ability of the endosomolytic ligand to promote endosomal escape, once the modular composition is taken up by the cell, can be evaluated, e.g., by preparation of an endosome preparation, or by microscopy techniques, which enable visualization of the labeled nucleic acid in the cytoplasm of the cell. In certain other

embodiments, the inhibition of gene expression, or any other physiological parameter, may be used as a surrogate marker for endosomal escape.

[00124] In other embodiments, circular dichroism spectroscopy can be used to identify compounds that exhibit a pH-dependent structural transition. [00125] A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to respond to changes in pH, and a second assay evaluates the ability of a modular composition that includes the test compound to respond to changes in pH.

[00126] Targeting Ligands [00127] The modular compositions of the present invention may comprise a targeting ligand. In some embodiments, this targeting ligand may direct the modular composition to, for example, a cell, cell type, tissue type or organ. For example, the targeting ligand may specifically or non-specifically bind with a molecule on the surface of a target cell. The targeting moiety can be a molecule with a specific affinity for a target cell. Targeting moieties can include antibodies directed against a protein found on the surface of a target cell, or the ligand or a receptor-binding portion of a ligand for a molecule found on the surface of a target cell. For example, the targeting moiety can recognize a cancer-specific antigen {e.g., CA15-3, CA19-9, CEA, or HER2/neu) or a viral antigen, thus delivering the polymer and a therapeutic agent, such as a nucleic acid associated with the polymer to a cancer cell or a virus-infected cell. Exemplary targeting moieties include antibodies of various isotypes (such as IgM, IgG, IgA, IgD, and the like, or a functional portion or fragment thereof), ligands for cell surface receptors (e.g.,

ectodomains thereof). Examples of fragments of antibodies include Fc, F(ab')₂, Fab' or the like.

[00128] Table 3 provides examples of a number of antigens - an antibody, or fragment thereof directed to such an antigen may be useful for selectively directing a polymer, or a composition coprising a polymer according to various embodiments of the invention.

[00129] Table 3. Exemplary antigens for targeting specific cells

ANTIGEN Exemplary tumor tissue

CEA (carcinoembryonic antigen) colon, breast, lung

PSA (prostate specific antigen) prostate cancer

CA-125 ovarian cancer

CA 15-3 breast cancer

CA 19-9 breast cancer

HER2/neu breast cancer

a-feto protein testicular cancer, hepatic cancer

β-HCG (human chorionic gonadotropin) testicular cancer, choriocarcinoma

MUC-1 breast cancer

Estrogen receptor breast cancer, uterine cancer

Progesterone receptor breast cancer, uterine cancer

EGFr (epidermal growth factor receptor) bladder cancer [00130] Ligand-mediated targeting to specific tissues through binding to their respective receptors on the cell surface may facilitate tissue-specific delivery of drugs. Specific targeting to disease-relevant cell types and tissues may allow for reduction of an effective dose, reduction of side effects, or improve the therapeutic index of the drug. Carbohydrates and carbohydrate clusters with multiple carbohydrate motifs are an example of targeting ligands, which may be used to target one or more drugs to particular tissues or cell types. For examples, see Hashida, M, Nishikawa, . et al. (2001) Cell-specific delivery of genes with glycosylated carriers. Adv. DrugDeliv. Rev. 52, 187-9; Monsigny, M., Roche, A.-C. et al. (1994). Glycoconjugates as carriers for specific delivery of therapeutic drugs and genes. Adv. Drug Deliv. Rev. 14, 1-24; Gabius, S., Kayser, K. et al. (1996). Endogenous lectins and neoglycoconjugates. A sweet approach to tumor diagnosis and targeted drug delivery. Eur. J. Pharm. and Biopharm. 42, 250- 261; Wadhwa, M. S., and Rice, K. G. (1995) Receptor mediated glycotargeting. J. Drug Target. 3, 111-127.

[00131] Another example of a receptor-ligand pair is the asialoglycoprotein receptor (ASGP-R), which is highly expressed on hepatocytes and which has a high affinity for D- galactose as well as N-acetyl-D-galactose (GalNAc). These carbohydrate ligands have demonstrated targeting of several drugs or carrier systems (e.g. liposomes, or polymeric carrier systems) to the liver parenchyma. For examples, see Wu, G. Y., and Wu, C. H. (1987) Receptor- mediated in vitro gene transformation by a soluble DNA carrier system. J. Biol. Chem. 262, 4429-4432; Biessen, E. A. L., Vietsch, H., Rump, E. T., Flutter, K., Bijsterbosch, M. K., and Van Berkel, T. J. C. (2000) Targeted delivery of antisense oligonucleotides to parenchymal liver cells in vivo. Methods Enzymol. 313, 324-342; Zanta, M.-A., Boussif, O., Adib, A., and Behr, J.-P. (1997) In Vitro Gene Delivery to Hepatocytes with Galactosylated Polyethylenimine.

Bioconjugate Chem. 8, 839-844; Managit, C, Kawakami, S. et al. (2003). Targeted and sustained drug delivery using PEGylated galactosylated liposomes. Int. J. Pharm. 266, 77-84; Sato, A., Takagi, M. et al. (2007). Small interfering RNA delivery to the liver by intravenous

administration of galactosylated cationic liposomes in mice. Biomaterials 28; 1434-42.

[00132] The mannose receptor, with its high affinity to D-mannose, is an example of carbohydrate-based ligand-receptor pair. The mannose receptor is expressed on, for example macrophages and dendritic cells. Mannose conjugates as well as mannosylated drug carriers have been successfully used to target drug molecules to those cells. For examples, see Biessen, E. A. L., Noorman, F. et al. (1996). Lysine-based cluster mannosides that inhibit ligand binding to the human mannose receptor at nanomolar concentration. J. Biol. Chem. 271, 28024-28030; Kinzel, O., Fattori, D.et al. (2003). Synthesis of a functionalized high affinity mannose receptor ligand and its application in the construction of peptide-, polyamide- and PNA-conjugates. J. Peptide Sci. 9, 375-385; Barratt, G., Tenu, J. P. et al. (1986). Preparation and characterization of liposomes containing mannosylated phospholipids capable of targeting drugs to macrophages. Biochim. Biophys. Acta 862, 153-64; Diebold, S. S., Plank, C. et al. (2002). Mannose Receptor- Mediated Gene Delivery into Antigen Presenting Dendritic Cells. Somat. Cell Mol. Genetics 27, 65-74.

[00133] Examples of carbohydrate-based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactose (GalNAc), multivalent GalNAc, e.g. GalNAC2 and GalNAc3; D-mannose, multivalent mannose, multivalent lactose, N-acetyl- galactosamine, N-acetyl-guIucosamine, multivalent fucose, glycosylated polyaminoacids and lectins. The term multivalent indicates that more than one monosaccharide unit is present. Such monosaccharide subunits may be linked to each other through glycosidic linkages or linked to a scaffold molecule.

[00134] Lipophilic moieties, such as cholesterol or fatty acids, when attached to highly hydrophilic molecules such as nucleic acids may enhance plasma protein binding and increase half-life of the nucleic acid in plasma, serum or blood. In addition, binding to certain plasma proteins, such as lipoproteins, has been shown to increase uptake in specific tissues expressing the corresponding lipoprotein receptors (e.g., LDL-receptor or the scavenger receptor SR-B1). For examples, see Bijsterbosch, M. K., Rump, E. T. et al. (2000). Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation. Nucleic Acids Res. 28, 2717-25; Wolfrum, C, Shi, S. et al. (2007). Mechanisms and optimization of in vivo delivery of lipophilic siR As. Nat. Biotechnol. 25, 1149-57. Lipophilic conjugates may therefore also be considered as a targeted delivery approach Movement of a polymer composition comprising a therapeutic agent within a cell (e.g. movement from the endosome to an organelle, another vesicle or to another intracellular location) may also be facilitated by combining the polymer composition with a targeting ligan, and intracellular trafficking may also be facilitated or enhanced in combination with endosomolytic ligands. [00135] Exemplary lipophilic moieties that may enhance plasma protein binding include, but are not limited to, sterols, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, adamantane acetic acid, 1- pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, boraeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytntyl, phenoxazine, aspirin, naproxen, ibuprofen, vitamin E and biotin etc.

[00136] Folates represent another class of ligands which has been widely used for targeted drug delivery via the folate receptor. This receptor is highly expressed on a wide variety of tumor cells, as well as other cells types, such as activated macrophages. For examples, see Matherly, L. H. and Goldman, I. D. (2003). Membrane transport of folates. Vitamins Hormones 66, 403-456; Sudimack, J. and Lee, R. J. (2000). Targeted drug delivery via the folate receptor. Adv. Drug Delivery Rev. 41, 147-162. Similar to carbohydrate-based ligands, folates have been shown to be capable of delivering a wide variety of drugs, including nucleic acids and even liposomal carriers. For examples, see Reddy, J. A., Dean, D. et al. (1999). Optimization of Folate-Conjugated Liposomal Vectors for Folate Receptor-Mediated Gene Therapy. J. Pharm. Sci. 88, 1 112-1 1 18; Lu, Y. and Low P. S. (2002). Folate-mediated delivery of macromolecular anticancer therapeutic agents. Adv. Drug Delivery Rev. 54, 675-693; Zhao, X. B. and Lee, R. J. (2004). Tumor-selective targeted delivery of genes and antisense oligodeoxyribonucleotides via the folate receptor; Leamon, C. P., Cooper, S. R. et al. (2003). Folate-Liposome-Mediated Antisense Oligodeoxynucleotide Targeting to Cancer Cells: Evaluation in Vitro and in Vivo. Bioconj. Chem. 14, 738-747.

[00137] United States Patent Publications No. US2009/0247614 and US2009/0239814 describe a number of folate and carbohydrate targeting ligands that may be amenable to the modular compositions of the present invention. Contents of these patent applications are herein incorporated by reference in their entirety. [00138] A targeting ligand may also be a protein, peptide or peptidomimetic that can target one or more cell markers, e.g. markers enriched in proliferating cells. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic moiety can be from about 5 to about50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long, or any amount therebetween. Such peptides include, but are not limited to, RGD-containing peptides and peptidomimetics that can target cancer cells, in particular cells that exhibit α_νβ₃ (alpha.v.beta.3) integrin. Targeting peptides can be linear or cyclic, and include D- amino acids, non-peptide or pseudo-peptide linkages, peptidyl mimics. In addition, the peptide and peptide mimics can be modified, e.g. glycosylated or methylated. Synthetic mimics of targeting peptides are also included. [00139] A targeting ligand can also include other receptor binding ligands such as hormones and hormone receptor binding ligands. For example, a targeting ligand can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin, glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, folate, vitamin B12, biotin, or an aptamer. Table 4 shows some examples of targeting ligands and their associated receptors.

[00140] Table 4: Liver Targeting Ligands and their associated receptors

[00141 ] When two or more targeting ligands are present, such targeting ligands may all be the same or different targeting ligands that target the same cell/tissue/organ. [00142] In addition to the endosomolytic ligand and the targeting ligand, the modular composition may comprise one or more other moieties/ligands that may enhance circulation half life and/or cellular uptake. These can include naturally occurring substances, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); or a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid). These moieties may also be a recombinant or synthetic molecule, such as a synthetic polymer or synthetic polyamino acids. Examples include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride polymer, poly(L-lactide-co-glycolied) polymer, divinyl ether-maleic anhydride polymer, N-(2- hydroxypropyl)methacrylamide polymer (HMPA), polyethylene glycol (PEG, e.g., PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), methyl-PEG (mPEG), [mPEG]₂, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide. [00143] Oligonucleotides and oligomeric compounds that comprise a number of phosphorothioate linkages are known in the art to bind to serum protein, thus short

oligonucleotides, e.g. oligonucleotides of about 5 to about 50 nucleotides, for example 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 bases or any amount therebetween. Non-nucleosidic oligomeric compounds comprising multiple phosphorothioate linkages can be used to enhance the circulation half life of the modular composition of the invention. In addition, oligonucleotides, e.g. aptamers, that bind serum ligands (e.g. serum proteins) can also be used to enhance the circulation half life of the modular composition of the invention. These oligonucleotides and aptamers may comprise any nucleic acid modification, e.g. sugar modification, backbone modification or nucleobase modification, described in this application. [00144] Compounds that increase the cellular uptake of the modular composition comprising one or more of formula I to V, or a combination thereof, may also be present in addition to the endosomolytic ligand and the targeting ligand. Exemplary compunds that enhance cellular uptake include vitamins. These are particularly useful for targeting

cells/tissues/organs characterized by unwanted cell proliferation, e.g., of the malignant or non- malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and . Other exemplary vitamins include B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells.

[00145] A compound that increases cellular uptake of a polymer of the present invenmtion can also be a substance, e.g, a drug, which can increase the uptake of the modular composition into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[00146] The compound can increase the uptake of the modular composition into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.

[00147] In some embodiments, such a compound is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the permeation agent is amphipathic. The helical cell permeation agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

[00148] Other compounds that can be present in the modular composition of the invention include, dyes and reporter groups for monitoring distribution, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating agents, phosphate, mercapto, amino, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles, dinitrophenyl, HRP and AP. [00149] In some embodiments, a single ligand may have more than one property, e.g. ligand has both endosomolytic and targeting properties.

[00150] Pharmacokinetic (PK) Modulators

Examples of pharmacokinetic (PK) modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g. oligonucleotides of about 5 to about 50 nucleotides, for example 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 bases or any amount therebetween, comprising multiple of phosphorothioate linkages in the backbaone are also amenable to the present invention as ligands (e.g. as P modulating ligands).

[00151] Enhanced Permeability and Retention

[00152] In certain embodiments, the modular composition of the invention may be targeted to a site via the enhanced permeability and retention (EPR) effect. The EPR effect is the property by which certain sizes of molecules, typically macromolecules, tend to accumulate in, for example, tumor tissue to a greater extent than in normal tissue. Without wishing to be bound by theory, the general explanation for this phenomenon is that the blood vessels supplying a tumor are typically abnormal in their architecture, containing wide fenestrations which permit the diffusion of macromolecules from the blood. Moreover, tumors typically lack effective lymphatic drainage, leading to the accumulation of molecules that diffuse from the blood. A person of ordinary skill in the art will recognize that such methods of targeting may also be useful for other conditions in which abnormal vasculature enable access to a specific site, with or without compromised lymphatic drainage. [00153] Representative United States patents that teach the preparation of oligonucleotide conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105;

5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731 ; 5,591,584;

5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046;

4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941 ; 4,835,263; 4,876,335;

4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,149,782;

5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;

5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;

5,567,810; 5,574, 142; 5,585,481 ; 5,587,371 ; 5,595,726; 5,597,696; 5,599,923; 5,599,928;

5,672,662; 5,688,941 ; 5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434; 6,335,437;

6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631 ; 6,559,279; each of which is herein incorporated by reference. [00154] Where more than one endosomolytic ligand or targeting ligand is present on the same modular composition, the more than one endosomolytic ligand or targeting ligand may be linked to the oligonucleotide strand or an endosomolytic ligand or targeting ligand in a linear fashion, or by a branched linker group. [00155] Cleavable Linker

[00156] A cleavable linker is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linker is cleaved at least 10 times or more, preferably at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

[00157] Cleavable linkers are susceptible to cleavage by , for example , pH, redox potential or the presence of degradative molecules including enzymes Generally, cleavage conditions or cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linker by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linker by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

[00158] A cleavable linker, such as a disulfide bond can be susceptible to pH (pH labile?). The pH of normal human serum is about 7.4, while the average, normal intracellular pH is slightly lower, ranging from about 7.1 -7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some spacer may have a linker that is cleaved at a preferred pH, thereby releasing the nucleic acid from the carrier oligomer inside the cell, or into the desired compartment of the cell.

[00159] A spacer can include a linker that is cleavable by a particular enzyme. The type of linker incorporated into a spacer can depend on the cell to be targeted by an nucleic acid. For example, an nucleic acid that targets an mRNA in liver cells can be linked to the carrier oligomer through a spacer that includes an ester group. Liver cells are rich in esterases, and therefore the spacer or tether will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Cleavage of the spacer releases the nucleic acid from the carrier oligomer, thereby potentially enhancing silencing activity of the nucleic acid. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

[00160] Spacers that contain peptide bonds can be used when the nucleic acids are targeting cell types rich in peptidases, such as liver cells and synoviocytes. For example, an nucleic acid targeted to synoviocytes, such as for the treatment of an inflammatory disease (e.g., rheumatoid arthritis), can be linked to a carrier oligomer through spacer that comprises a peptide bond.

[00161] In general, the suitability of a cleavable linker can be evaluated by testing the ability of a degradative agent (or condition) to cleave the linker. It will also be desirable to also test the cleavable linker for the ability to resist cleavage in the blood or when in contact with other non-target tissue, e.g., tissue the nucleic acid would be exposed to when administered to a subject. Thus one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. Initial evaluations in cell-free or culture conditions may be confirmed by further evaluations in whole animals. In some embodiments, compounds (e.g. a spacrer, a linker ) are those that are cleaved at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions). [00162] Redox cleavable linkers

[001 3] One class of cleavable linkers are redox cleavable linkers that are cleaved upon reduction or oxidation. An example of reductively cleavable linker is a disulphide linker (-S-S-). To determine if a candidate cleavable linker is a suitable "reductively cleavable linker," or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In a preferred embodiment, candidate compounds are cleaved by at most 10% in the blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media. [00164] Phosphate-based cleavable linkers

[00165] Phosphate-based linkers are cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linkers are -0-P(0)(ORk)-0-, -O- P(S)(ORk)-0-, -0-P(S)(SRk)-0-, -S-P(0)(ORk)-0-, -0-P(0)(ORk)-S-, -S-P(0)(ORk)-S-, -O- P(S)(ORk)-S-, -S-P(S)(ORk)-0-, -0-P(0)(Rk)-0-, -0-P(S)(Rk)-0-, -S-P(0)(Rk)-0-, -S- P(S)(Rk)-0-, -S-P(0)(Rk)-S-, -0-P(S)( Rk)-S-. Some exemplary embodiments are -O- P(0)(OH)-0-, -0-P(S)(OH)-0-, -0-P(S)(SH)-0-, -S-P(0)(OH)-0-, -0-P(0)(OH)-S-, -S- P(0)(OH)-S-, -0-P(S)(OH)-S-, -S-P(S)(OH)-0-, -0-Ρ(0)(Η)-0-, -0-P(S)(H)-0-, -S-P(0)(H)-0-, -S-P(S)(H)-0-, -S-P(0)(H)-S-, -0-P(S)(H)-S-. An exemplary embodiment is -0-P(0)(OH)-0-. These candidates can be evaluated using methods analogous to those described above.

[00166] Acid cleavable linkers

[00167] Acid cleavable linkers are linkers that are cleaved under acidic conditions. In exemplary embodiments, acid cleavable linkers may be cleaved in an acidic environment with a pH of about 6.5 or lower, for example from 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5 or 1.0, or any amount therebetween, or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linkers. Examples of acid cleavable linkers include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C=NN-, C(0)0, or -OC(O). An exemplary embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t These candidates can be evaluated using methods analogous to those described above.

[00168] Ester-based linkers

[00169] Ester-based linkers are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linkers include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linkers have the general formula -C(0)0-, or -OC(O)-. These candidates can be evaluated using methods analogous to those described above.

[00170] Peptide-based cleaving groups

[00171] Peptide-based linkers are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linkers are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide cleavable linkers have the general formula - NHCHR³C(0)NHCHR⁴C(0)-, where R³ and R⁴ are the side chain groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above. [00172] iRNA Asents

[00173] iRNA agents are examples of nucleic acids. Generally, an iRNA agent comprises a region of sufficient homology to allow for specific hybridization to a transcript of a target gene, and be of sufficient length in terms of nucleotides, such that the iRNA agent, or a fragment thereof, can modulate expression of the target gene product. (For ease of exposition the term nucleotide or ribonucleotide is sometimes used herein in reference to one or more monomelic subunits of an RNA agent. It will be understood herein that the usage of the term

"ribonucleotide" or "nucleotide", herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.) Thus, the iRNA agent is or includes a region which is at least partially, and in some embodiments fully, complementary to the target RNA. It is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA. Complementarity, or degree of homology with the target strand, is most critical in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired some embodiments can include, particularly in the antisense strand, one or more, or for example, 6, 5, 4, 3, 2, or fewer mismatches (with respect to the target RNA). The mismatches, particularly in the antisense strand, are most tolerated in the terminal regions and if present may be in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5' and/or 3' termini. The sense strand need only be sufficiently complementary with the antisense strand to maintain the over all double stranded character of the molecule.

[00174] As discussed elsewhere herein, and in the material incorporated by reference in its entirety, an iRNA agent will often be modified or include nucleoside surrogates. Single stranded regions of an iRNA agent will often be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates. Modification to stabilize one or more 3'- or 5'-termini of an iRNA agent, e.g., against exonucleases, or to favor the antisense siRNA agent to enter into RISC are also envisioned. Modifications can include C3 (or C6, C7, CI 2) amino linkers, thiol linkers, carboxyl linkers, non-nucleotide spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis.

[00175] iRNA agents include molecules that are long enough to trigger the interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencing complex)); and, molecules which are sufficiently short that they do not trigger the interferon response (which molecules can also be cleaved by Dicer and/or enter a RISC), e.g., molecules which are of a size which allows entry into a RISC, e.g., molecules which resemble Dicer-cleavage products. Molecules that are short enough that they do not trigger an interferon response are termed siRNA agents or shorter iRNA agents herein. "siRNA agent or shorter iRNA agent" as used herein, refers to an iRNA agent, e.g., a double stranded RNA agent or single strand agent, that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60, 50, 40, or 30 nucleotide pairs. The siRNA agent, or a cleavage product thereof, can down regulate a target gene, e.g., by inducing RNAi with respect to a target RNA, wherein the target may comprise an endogenous or pathogen target RNA.

[00 76] Each strand of an siRNA agent may be from about 10 to about 30 nucleotides in length, or any amount therebetween , for example about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. The strand may be at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. siRNA agents may have a duplex region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotide pairs, and one or more overhangs, or one or two 3 ' overhangs, of 2- 3 nucleotides.

[00177] In addition to homology to target RNA and the ability to down regulate a target gene, an iRNA agent may have one or more of the following properties:

(1) if single stranded it may have a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group; (2) it may, despite modifications, even to a very large number, or all of the nucleosides, have an antisense strand that can present bases (or modified bases) in the proper three

dimensional framework so as to be able to form correct base pairing and form a duplex structure with a homologous target RNA which is sufficient to allow down regulation of the target, e.g., by cleavage of the target RNA;

(3) it may, despite modifications, even to a very large number, or all of the nucleosides, still have "RNA-like" properties, i.e., it may possess the overall structural, chemical and physical properties of an RNA molecule, even though not exclusively, or even partly, of ribonucleotide- based content. For example, an iRNA agent can contain, e.g., a sense and/or an antisense strand in which all of the nucleotide sugars contain e.g., 2' fluoro in place of 2' hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties.

While not wishing to be bound by theory, the electronegative fluorine prefers an axial orientation when attached to the C2' position of ribose. This spatial preference of fluorine can, in turn, force the sugars to adopt a Cy-endo pucker. This is the same puckering mode as observed in RNA molecules and gives rise to the RNA-characteristic A-family-type helix.

Further, since fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bonding interactions with water molecules that are known to stabilize RNA structures. A modified moiety at the 2' sugar position may be able to enter into H bonding which is more characteristic of the OH moiety of a ribonucleotide than the H moiety of a deoxyribonucleotide. Certain iRNA agents will: exhibit a Cy-endo pucker in all, or at least 50, 75,80, 85, 90, or 95 % of its sugars; exhibit a Cy-endo pucker in a sufficient amount of its sugars that it can give rise to a the RNA-characteristic A-family-type helix; will have no more than 20, 10, 5, 4, 3, 2, orl sugar which is not a Cy-endo pucker structure. Regardless of the nature of the modification, and even though the RNA agent can contain deoxynucleotides or modified deoxynucleotides, particularly in overhang or other single strand regions, it is certain DNA molecules, or any molecule in which more than 50, 60, or 70 % of the nucleotides in the molecule, or more than 50, 60, or 70 % of the nucleotides in a duplexed region are deoxyribonucleotides, or modified deoxyribonucleotides which are deoxy at the 2' position, are excluded from the definition of RNA agent.

[00178] A "single strand iRNA agent" as used herein, is an iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin or pan-handle structure. Single strand iRNA agents may be antisense with regard to the target molecule. In certain embodiments single strand iRNA agents are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus. 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5 '-monophosphate ((HO)2(0)P-0-5'); 5'-diphosphate ((HO)2(0)P-0- P(HO)(0)-0-5'); 5'-triphosphate ((HO)2(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-0-5"-(HO)(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'); 5'- adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'- (HO)(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'); 5'-monothiophosphate (phosphorothioate;

(HO)2(S)P-0-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-0-5'), 5'- phosphorothiolate ((HO)2(0)P-S-5'); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma- thiotriphosphate, etc.), 5'-phosphoramidates ((ΗΟ)2(0)Ρ-ΝΗ-5', (ΗΟ)(ΝΗ2)(0)Ρ-0-5'), 5'- alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(0)-0-5'-, (OH)2(0)P-5'-CH2-), 5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(0)-0-5'-). (These modifications can also be used with the antisense strand of a double stranded iRNA.) [00179] A single strand iRNA agent may be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA. A single strand iRNA agent may be from about 10 to about 200 nucleotides in length, or any amount therebetween, or about 10, 1 1, 12, 13, 14„ 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200 nucleotides in length, or any amount therebetween.

[00180] Hairpin iRNA agents comprise a duplex region of about 10 to about 30 base pairs in length, or any amount therebetween, or about 10, 11, 12, 13, 14„ 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 base pairs in length, or any amount therebetween. The duplex region will may be equal to or less than 200, 100, or 50, in length. Particular exemplary embodiments include 17 to 25, 19 to 23, or 19 to21 base pairs in length. The hairpin may have a single strand overhang or terminal unpaired region, in some embodiments at the 3', and in certain embodiments on the antisense side of the hairpin. In some embodiments, the overhangs are 2-3 nucleotides in length.

[00181 ] A "double stranded (ds) iRNA agent" as used herein, is an iRNA agent which includes more than one, and in some cases two, strands in which interchain hybridization can form a region of duplex structure.

[00182] An antisense strand of a double stranded iRNA agent may be from about 10 to about 200 nucleotides in length, or any amount therebetween, or about 10, 11, 12, 13, 14„ 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200 nucleotides in length, or any amount therebetween Particular exemplary

embodiments include 17 to 25, 19 to 23, or 19 to21 nucleotides in length.

[00183] The sense strand of a double stranded iRNA agent may be may be from about 10 to about 200 nucleotides in length, or any amount therebetween, or about 10, 1 1, 12, 13, 14„ 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200 nucleotides in length, or any amount therebetween. Particular exemplary embodiments include 17 to 25, 19 to 23, or 19 to21 nucleotides in length.

[00184] The double strand portion of a double stranded iRNA agent may be from about 10 to about 200 nucleotides in length, or any amount therebetween, or about 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200 nucleotides in length, or any amount therebetween. Particular exemplary embodiments include 17 to 25, 19 to 23, or 19 to 21 nucleotides in length.

[00185] In many embodiments, the ds iRNA agent may be sufficiently large that it can cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller ds iRNA agents, e.g., siRNAs agents.

[00186] The present invention further includes iRNA agents that target within the sequence targeted by one of the iRNA agents of the present invention. As used herein a second iRNA agent is said to target within the sequence of a first iRNA agent if the second iRNA agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first iRNA agent. Such a second agent will generally consist of at least 15 contiguous nucleotides coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in the target gene.

[00187] The dsiRNAs of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsiRNA of the invention contains no more than 3 mismatches. If the antisense strand of the dsiRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of

complementarity. If the antisense strand of the dsiRNA contains mismatches to the target sequence, the mismatch may be found within 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5' or 3' end of the region of complementarity. For example, for a 23 nucleotide dsiRNA strand which is complementary to a region of the target gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsiRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the target gene.

Consideration of the efficacy of dsiRNAs with mismatches in inhibiting expression of the target gene may be important, especially if the particular region of complementarity in the target gene is known to have polymorphic sequence variation within the population.

[00188] In some embodiments, the sense-strand comprises a mismatch to the antisense strand. In some embodiments, the mismatch may be fund within 5 nucleotides from the 3 '-end, for example 5, 4, 3, 2, or 1 nucleotides from the end of the region of complementarity. In some embodiments, the mismatch is located in the target cleavage site region. In one₌embodiment, the sense strand comprises no more than 1, 2, 3, 4 or 5 mismatches to the antisense strand. In some embodiments, the sense strand comprises no more than 3 mismatches to the antisense strand.

[00189] In certain embodiments, the sense strand comprises a nucleobase modification, e.g. an optionally substituted natural or non-natural nucleobase, a universal nucleobase, in the target cleavage site region.

[00190] The phrase "target cleavage site" as used herein refers to the backbone linkage in the target gene, e.g. target mRNA, or the sense strand that is cleaved by the RISC mechanism by utilizing the iRNA agent. And the "target cleavage site region" comprises at least one or at least two nucleotides on both side of the cleavage site. As an example, the target cleavage site on a sense strand of a double-stranded nucleic acid is the backbone linkage in the sense strand that would get cleaved if the sense strand itself was the target to be cleaved by the RNAi mechanism. The target cleavage site can be determined using methods known in the art, for example the 5'- RACE assay as detailed in Soutschek et al., Nature (2004) 432, 173-178. As is well understood in the art, the cleavage site region for a conical double stranded RNAi agent comprising two 21- nucleotides long strands (wherin the strands form a double stranded region of 19 consective basepairs having 2-nucleotide single stranded overhangs at the 3 '-ends), the cleavage site region corresponds to postions 9-12 from the 5 '-end of the sense strand.

[00191] The present invention also includes nucleic acids which are chimeric compounds. "Chimeric" nucleic acid compounds or "chimeras," in the context of this invention, are nucleic acid compounds, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a nucleic acid compound. These nucleic acids typically contain at least one region wherein the nucleic acid is modified so as to confer upon the it increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the nucleic acid may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an

RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression.

[00192] The present invention also includes ds iRNAs wherein the two strands are linked together. The two strands can be linked together by a polynucleotide linker such as (dT)_n;

wherein n is 4-10, and thus forming a hairpin. The two strands can also be linked together by a non-nucleosidic linker, e.g. a linker described herein. It will be appreciated by one of skill in the art that any oligonucleotide chemical modifications or variations describe herein can be used in the polynucleotide linker.

[00193] The double stranded oligonucleotides can be optimized for RNA interference by increasing the propensity of the duplex to disassociate or melt (decreasing the free energy of duplex association), in the region of the 5' end of the antisense strand This can be

accomplished, e.g., by the inclusion of modifications or modified nucleosides which increase the propensity of the duplex to disassociate or melt in the region of the 5' end of the antisense strand. It can also be accomplished by inclusion of modifications or modified nucleosides or attachment of a ligand that increases the propensity of the duplex to disassociate of melt in the region of the 5 'end of the antisense strand. While not wishing to be bound by theory, the effect may be due to promoting the effect of an enzyme such as helicase, for example, promoting the effect of the enzyme in the proximity of the 5' end of the antisense strand.

[00194] Definitions [00195] The term "random polymer" as used herein refers to a polymer in which the sequential distribution of the monomelic units obeys known statistical laws, e.g. the sequential distribution of monomer units follows Markovian statistics.

[00196] The term "block polymer" as used herein refers to a polymer comprising of macromolecules consisting of a linear sequence of blocks, wherein the term "block" means a portion of macromolecule comprising many constitutional units that has at least one feature that is not present in the adjacent portions.

[00197] The term "polymer matrix" as used herein refers to tone or more polymer layers or sublayers on the metal surface. This can include activating, first, additional, and/or barrier layers.

[00198] The term "amphiphilic polymer" as used herein refers to a polymer containing both hydrophilic (water-soluble) and hydrophobic (water-insoluble) segments.

[00199] The terms "silence" and "inhibit the expression of and related terms and phrases, refer to the at least partial suppression of the expression of a gene targeted by an siRNA or short interfering nucleic acid (siNA). Silencing of a gene may be observed as a reduction of the amount of mRNA or other RNA molecule transcribed from the target gene, or in a reduction of the polypeptide encoded by the mRNA. The RNA or polypeptide may be isolated from a first cell or group of cells in which the target gene is expected to be transcribed or expressed and which has or have been treated such that the transcription or expression of the target gene is inhibited (e.g. by contacting the cell with a nucleic acid such as an antisense, siRNA or iRNA), as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (i.e., control cells).

[00200] The term "halo" or "halogen" as used herein refers to any radical of fluorine, chlorine, bromine or iodine.

[00201 ] The term "aliphatic," as used herein, refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, or from 1 to about 12 carbon atoms, or from 1 to about 6 carbon atoms, for example 1, 2, 3, 4,5, 6, 7, 8,9, 0, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substitutent groups.

[00202] The term "acyl" as used herein refers to hydrogen, alkyl, partially saturated or fully saturated cycloalkyl, partially saturated or fully saturated heterocycle, aryl, heteroaryl substituted carbonyl groups or the like. For example, acyl includes groups such as (Ci- C6)alkanoyl (e.g., formyl, acetyl, propionyl, butyryl, valeryl, caproyl, t- butylacetyl, etc.), (C3- Ce)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl, pyrrolid-2-one-5 - carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl) and heteroaroyl (e.g., thiophenyl-2-carbonyl, thiophenyl-3 -carbonyl, furanyl-2- carbonyl, furanyl-3 -carbonyl, lH-pyrroyl-2-carbonyl, lH-pyrroyl-3 -carbonyl,

benzo[b]thiophenyl-2-carbonyl, etc.). In addition, the alkyl, cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl group may be any one of the groups described in the respective definitions. When indicated as being "optionally substituted", the acyl group may be

unsubstituted or optionally substituted with one or more substituents (typically, one to three substituents) independently selected from the group of substituents listed below in the definition for "substituted" or the alkyl, cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl group may be substituted as described above in the preferred and more preferred list of substituents, respectively. [00203] For simplicity, chemical moieties are defined and referred to throughout can be univalent chemical moieties (e.g., alkyl, aryl, etc.) or multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, an "alkyl" moiety can be referred to a monovalent radical (e.g. CH3-CH2-), or in other instances, a bivalent linking moiety can be "alkyl," in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., -CH2-CH2-), which is equivalent to the term "alkylene." Similarly, in circumstances in which divalent moieties are required and are stated as being "alkoxy", "alkylamino", "aryloxy", "alkylthio", "aiyl", "heteroaryl", "heterocyclic", "alkyl" "alkenyl", "alkynyl", "aliphatic", or "cycloalkyl", those skilled in the art will understand that the terms alkoxy", "alkylamino", "aryloxy", "alkylthio", "aryl", "heteroaryl", "heterocyclic", "alkyl", "alkenyl", "alkynyl",

"aliphatic", or "cycloalkyl" refer to the corresponding divalent moiety.

[00204] The term "alkyl" refers to saturated and unsaturated non-aromatic hydrocarbon chains that may be a straight chain or branched chain, containing the indicated number of carbon atoms (these include without limitation propyl, allyl, or propargyl), which may be optionally inserted with N, O, or S. For example, CpCio indicates that the group may have from 1 to 10 (inclusive) carbon atoms in it. The term "alkoxy" refers to an -O-alkyl radical. The term

"alkylene" refers to a divalent alkyl (i.e., -R-). The term "alkyl enedioxo" refers to a divalent species of the structure -O-R-O-, in which R represents an alkylene. The term "aminoalkyl" refers to an alkyl substituted with an amino. The term "mercapto" refers to an -SH radical. The term "thioalkoxy" refers to an -S-alkyl radical. [00205] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1 , 2, 3, or 4 atoms of each ring may be substituted by a substituent.

Examples of aryl groups include phenyl, naphthyl and the like. The term "arylalkyl" or the term "aralkyl" refers to alkyl substituted with an aryl. The term "arylalkoxy" refers to an alkoxy substituted with aryl.

[00206] The term "cycloalkyl" as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, wherein the cycloalkyl group additionally may be optionally substituted. Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[00207] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11 - 14 membered tricyclic ring system having 1 -3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, the heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1 , 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like. The term "heteroarylalkyl" or the term "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with heteroaryl.

[00208] The term "heterocyclyl" or "heterocyclic" refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, the heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1 , 2 or 3 atoms of each ring may be substituted by a substituent. Examples of heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like. [00209] The term "substituents" refers to a group "substituted" on an alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of that group. Suitable substituents include, without limitation, halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, ureido or conjugate groups.

[00210] In many cases, protecting groups are used during preparation of the compounds of the invention. As used herein, the term "protected" means that the indicated moiety has a protecting group appended thereon. In some preferred embodiments of the invention, compounds contain one or more protecting groups. A wide variety of protecting groups can be employed in the methods of the invention. In general, protecting groups render chemical functionalities inert to specific reaction conditions, and can be appended to and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule.

[00211] Representative hydroxyl protecting groups, for example, are disclosed by

Beaucage et al. (Tetrahedron 1992, 48, 2223-2311). Further hydroxyl protecting groups, as well as other representative protecting groups, are disclosed in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, and

Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y, 1991.

[00212] Examples of hydroxyl protecting groups include, but are not limited to, t-butyl, t- butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, l-(2-chloroethoxy)ethyl, 2- trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl,

diphenylmethyl, ρ,ρ'-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate, chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate, p-phenylbenzoate, 9- fluorenylmethyl carbonate, mesylate and tosylate. [00213] Nucleic acids

[00214] MicroRNAs

[00215] MicroRNAs (miRNAs or mirs) are a highly conserved class of small RNA molecules that are transcribed from DNA in the genomes of plants and animals, but are not translated into protein. Pre-microRNAs are processed into miRNAs. Processed microRNAs are single stranded—17-25 nucleotide (nt) RNA molecules that become incorporated into the RNA- induced silencing complex (RISC) and have been identified as key regulators of development, cell proliferation, apoptosis and differentiation. They are believed to play a role in regulation of gene expression by binding to the 3 '-untranslated region of specific mRNAs. RISC mediates down-regulation of gene expression through translational inhibition, transcript cleavage, or both. RISC is also implicated in transcriptional silencing in the nucleus of a wide range of eukaryotes.

[00216] The number of miRNA sequences identified to date is large and growing, illustrative examples of which can be found, for example, in: "miRBase: microRNA sequences, targets and gene nomenclature" Griffiths- Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. NAR, 2006, 34, Database Issue, D140-D144; "The microRNA Registry" Griffiths- Jones S. NAR, 2004, 32, Database Issue, D109-D111.

[00217] Single-stranded oligonucleotides, including those described and/or identified as microRNAs or mirs which may be used as targets or may serve as a template for the design of oligonucleotides of the invention are taught in, for example, Esau, et al. US Publication No. 2005/0261218 entitled "Oligomeric compounds and compositions for use in modulation small non-coding RNAs" the entire contents of which is incorporated herein by reference. It will be appreciated by one of skill in the art that any oligonucleotide chemical modifications or variations describe herein also apply to single stranded oligonucleotides. [00218] miRNA mimics

[00219] miRNA mimics represent a class of molecules that can be used to imitate the gene silencing ability of one or more miRNAs. Thus, the term "microRNA mimic" refers to synthetic non-coding RNAs (i.e. the miRNA is not obtained by purification from a source of the endogenous miRNA) that are capable of entering the RNAi pathway and regulating gene expression. miRNA mimics can be designed as mature molecules (e.g. single stranded) or mimic precursors (e.g., pri- or pre-miRNAs). miRNA mimics can be comprised of nucleic acid

(modified or modified nucleic acids) including oligonucleotides comprising, without limitation, RNA, modified RNA, DNA, modified DNA, locked nucleic acids, or 2'-0,4'-C-ethylene-bridged nucleic acids (ENA), or any combination of the above (including DNA-RNA hybrids). In addition, miRNA mimics can comprise conjugates that can affect delivery, intracellular compartmentalization, stability, specificity, functionality, strand usage, and/or potency. In one design, miRNA mimics are double stranded molecules (e.g., with a duplex region of between about 16 and about 31 nucleotides in length) and contain one or more sequences that have identity with the mature strand of a given miRNA. Modifications can comprise 2' modifications (including 2'-0 methyl modifications and 2' F modifications) on one or both strands of the molecule and internucleotide modifications (e.g. phorphorthioate modifications) that enhance nucleic acid stability and/or specificity. In addition, miRNA mimics can include overhangs. The overhangs can consist of 1-6 nucleotides on either the 3' or 5' end of either strand and can be modified to enhance stability or functionality. In one embodiment, a miRNA mimic comprises a duplex region of between 16 and 31 nucleotides and one or more of the following chemical modification patterns: the sense strand contains 2'-0-methyl modifications of nucleotides 1 and 2 (counting from the 5' end of the sense oligonucleotide), and all of the Cs and Us; the antisense strand modifications can comprise 2' F modification of all of the Cs and Us, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleotide linkages associated with a 2 nucleotide 3 ' overhang. [00220] Supermirs

[00221] A supermir refers to a single stranded, double stranded or partially double stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both or modifications thereof, which has a nucleotide sequence that is substantially identical to an miRNA and that is antisense with respect to its target. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intemucleoside (backbone) linkages and which contain at least one non-naturally-occurring portion which functions similarly. Such modified or substituted oligonucleotides are preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. In a preferred

embodiment, the supermir does not include a sense strand, and in another preferred embodiment, the supermir does not self-hybridize to a significant extent. A supermir featured in the invention can have secondary structure, but it is substantially single-stranded under physiological conditions. An supermir that is substantially single-stranded is single-stranded to the extent that less than about 50% (e.g., less than about 40%, 30%, 20%, 10%, or 5%) of the supermir is duplexed with itself. The supermir can include a hairpin segment, e.g., sequence, preferably at the 3' end can self hybridize and form a duplex region, e.g., a duplex region of at least 1, 2, 3, or 4 and preferably less than 8, 7, 6, or n nucleotides, e.g., 5 nuclotides. The duplexed region can be connected by a linker, e.g., a nucleotide linker, e.g., 3, 4, 5, or 6 dTs, e.g., modified dTs. In another embodiment the supermir is duplexed with a shorter oligo, e.g., of 5, 6, 7, 8, 9, or 10 nucleotides in length, e.g., at one or both of the 3' and 5' end or at one end and in the nonterminal or middle of the supermir.

[00222] Antimir or miRNA inhibitor

[00223] The terms " antimir" "microRNA inhibitor", "miR inhibitor", or "inhibitor" are synonymous and refer to oligonucleotides or modified oligonucleotides that interfere with the ability of specific miRNAs. In general, the inhibitors are nucleic acid or modified nucleic acids in nature including oligonucleotides comprising RNA, modified RNA, DNA, modified DNA, locked nucleic acids (LNAs), or any combination of the above. Modifications include 2' modifications (including 2'-0 alkyl modifications and 2' F modifications) and internucleotide modifications (e.g. phosphorothioate modifications) that can affect delivery, stability, specificity, intracellular compartmentalization, or potency. In addition, miRNA inhibitors can comprise conjugates that can affect delivery, intracellular compartmentalization, stability, and/or potency. Inhibitors can adopt a variety of configurations including single stranded, double stranded (RNA/RNA or RNA/DNA duplexes), and hairpin designs, in general, microRNA inhibitors comprise contain one or more sequences or portions of sequences that are complementary or partially complementary with the mature strand (or strands) of the miRNA to be targeted, in addition, the miRNA inhibitor may also comprise additional sequences located 5' and 3' to the sequence that is the reverse complement of the mature miRNA. The additional sequences may be the reverse complements of the sequences that are adjacent to the mature miRNA in the pri- miRNA from which the mature miRNA is derived, or the additional sequences may be arbitrary sequences (having a mixture of A, G, C, or U). In some embodiments, one or both of the additional sequences are arbitrary sequences capable of forming hairpins. Thus, in some embodiments, the sequence that is the reverse complement of the miRNA is flanked on the 5' side and on the 3' side by hairpin structures. Micro-RNA inhibitors, when double stranded, may include mismatches between nucleotides on opposite strands. Furthermore, micro-RNA inhibitors may be linked to conjugate moieties in order to facilitate uptake of the inhibitor into a cell. For example, a micro-RNA inhibitor may be linked to cholesteryl 5-(bis(4- methoxyphenyl)(phenyl)methoxy)-3 hydroxypentylcarbamate) which allows passive uptake of a micro-RNA inhibitor into a cell. Micro-RNA inhibitors, including hairpin miRNA inhibitors, are described in detail in Vermeulen et al., "Double-Stranded Regions Are Essential Design

Components Of Potent Inhibitors of RISC Function," RNA 13: 723-730 (2007) and in

WO2007/095387 and WO 2008/036825 each of which is incorporated herein by reference in its entirety. A person of ordinary skill in the art can select a sequence from the database for a desired miRNA and design an inhibitor useful for the methods disclosed herein.

[00224] Ul adaptors

[00225] Ul adaptors inhibit polyA sites and are bifunctional oligonucleotides with a target domain complementary to a site in the target gene's terminal exon and a 'Ul domain' that binds to the Ul smaller nuclear RNA component of the Ul snRNP (Goraczniak, et al., 2008, Nature Biotechnology, 27(3), 257-263, which is expressly incorporated by reference herein, in its entirety). Ul snRNP is a ribonucleoprotein complex that functions primarily to direct early steps in spliceosome formation by binding to the pre-mRNA exon- intron boundary (Brown and Simpson, 1998, Annu Rev Plant Physiol Plant Mol Biol 49:77-95). Nucleotides 2-11 of the 5'end of Ul snRNA base pair bind with the 5'ss of the pre mRNA. In one embodiment,

oligonucleotides of the invention are Ul adaptors. In one embodiment, the Ul adaptor can be administered in combination with at least one other iRNA agent.

[00226] Antagomirs

[00227] Antagomirs are RNA-like oligonucleotides that harbor various modifications for RNAse protection and pharmacologic properties, such as enhanced tissue and cellular uptake. They differ from normal RNA by, for example, complete 2'-O-methylation of sugar,

phosphorothioate backbone and, for example, a cholesterol-moiety at 3 '-end. Antagomirs may be used to efficiently silence endogenous miRNAs by forming duplexes comprising the antagomir and endogenous miRNA, thereby preventing miRNA-induced gene silencing. An example of antagomir-mediated miRNA silencing is the silencing of miR-122, described in Krutzfeldt et al, Nature, 2005, 438: 685-689, which is expressly incorporated by reference herein, in its entirety. Antagomir RNAs may be synthesized using standard solid phase oligonucleotide synthesis protocols. See, for example, US Patent Publication Nos. US 2007/0123482 and US

2007/0213292; the disclosure of each of which are incorporated herein by reference. An antagomir can include ligand-conjugated monomer subunits and monomers for oligonucleotide synthesis. Exemplary monomers are described in U.S. Patent Publication Nos US 2005-

0107325. An antagomir can have a ZXY structure, such as is described in PCT Application No. PCT/US2004/07070 filed on March 8, 2004. An antagomir can be complexed with an amphipathic moiety. Exemplary amphipathic moieties for use with oligonucleotide agents are described in PCT Publication No. WO/2004/080406. [00228] Antagomirs may be single stranded, double stranded, partially double stranded or hairpin-structured, chemically modified oligonucleotides that target a microRNA. An antagomir may consist essentially of or comprise about 12 or more contiguous nucleotides substantially complementary to an endogenous miRNA, and more particularly, agents that include about 12 or more contiguous nucleotides substantially complementary to a target sequence of an miRNA or pre-miRNA nucleotide sequence. In certain embodiments, an antagomir featured in the invention includes a nucleotide sequence sufficiently complementary to hybridize to a miRNA target sequence of about 12 to 25 nucleotides, in some instances about 15 to 23 nucleotides.

[00229] Decoy Oligonucleotides

[00230] Because transcription factors can recognize their relatively short binding sequences, even in the absence of surrounding genomic DNA, short oligonucleotides bearing the consensus binding sequence of a specific transcription factor can be used as tools for

manipulating gene expression in living cells. This strategy involves the intracellular delivery of such "decoy oligonucleotides", which are then recognized and bound by the target factor.

Occupation of the transcription factor's DNA-binding site by the decoy renders the transcription factor incapable of subsequently binding to the promoter regions of target genes. Decoys can be used as therapeutic agents, either to inhibit the expression of genes that are activated by a transcription factor, or to upregulate genes that are suppressed by the binding of a transcription factor. Examples of the utilization of decoy oligonucleotides may be found in Mann et al., J. Clin. Invest., 2000, 106: 1071-1075, which is expressly incorporated by reference herein, in its entirety.

[00231] An oligonucleotide agent featured in the invention can also be a decoy nucleic acid, e.g., a decoy RNA. A decoy nucleic acid resembles a natural nucleic acid, but may be modified in such a way as to inhibit or interrupt the activity of the natural nucleic acid. For example, a decoy RNA can mimic the natural binding domain for a ligand. The decoy RNA, therefore, competes with natural binding domain for the binding of a specific ligand. The natural binding target can be an endogenous nucleic acid, e.g., a pre-miR A, miRNA, pre-mRNA, mRNA or DNA. For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a "decoy" and efficiently bind HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA. In certain embodiments, a decoy RNA may include a modification that improves targeting, e.g., a targeting modification described herein.

[00232] Antisense Oligonucleotides

[00233] Antisense oligonucleotides are single strands of DNA or RNA that are at least partially complementary to a chosen sequence. In the case of antisense RNA, they prevent translation of complementary RNA strands by binding to it. Antisense DNA can also be used to target a specific, complementary (coding or non-coding) RNA. If binding takes place, the DNA/RNA hybrid can be degraded by the enzyme RNase H. Examples of the utilization of antisense oligonucleotides may be found in Dias et al., Mol. Cancer Ther., 2002, 1 : 347-355, which is expressly incorporated by reference herein, in its entirety. [00234] The single-stranded oligonucleotide agents featured in the invention include antisense nucleic acids. An "antisense" nucleic acid includes a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a gene expression product, e.g.,

complementary to the coding strand of a double-stranded cDNA molecule or complementary to an RNA sequence, e.g., a pre-mRNA, mRNA, miRNA, or pre-miRNA. Accordingly, an antisense nucleic acid may form hydrogen bonds with a sense nucleic acid target.

[00235] Given a coding strand sequence (e.g., the sequence of a sense strand of a cDNA molecule), antisense nucleic acids can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to a portion of the coding or noncoding region of an RNA, e.g., a pre-mRNA or mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a pre- mRNA or mRNA, e.g., the 5' UTR. An antisense oligonucleotide can be, for example, about 10 to 25 nucleotides in length or any amount therebetween, (e.g., about 1 1, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length). An antisense oligonucleotide can also be complementary to a miRNA or pre-miRNA. [00236] In certain embodiments, an antisense nucleic acid can be constructed using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and target nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Other appropriate nucleic acid modifications are described herein. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest). [00237] An antisense agent can include ribonucleotides only, deoxyribonucleotides only (e.g., oligodeoxynucleotides), or both deoxyribonucleotides and ribonucleotides. For example, an antisense agent consisting only of ribonucleotides can hybridize to a complementary RNA, and prevent access of the translation machinery to the target RNA transcript, thereby preventing protein synthesis. An antisense molecule including only deoxyribonucleotides, or

deoxyribonucleotides and ribonucleotides, e.g., DNA sequence flanked by RNA sequence at the 5' and 3' ends of the antisense agent, can hybridize to a complementary RNA, and the RNA target can be subsequently cleaved by an enzyme, e.g., RNAse H. Degradation of the target RNA prevents translation. The flanking RNA sequences can include 2'-0-methylated nucleotides, and phosphorothioate linkages, and the internal DNA sequence can include phosphorothioate internucleotide linkages. In some embodiments, the internal DNA sequence may be at least five nucleotides in length when targeting by RNAseH activity is desired.

[00238] For increased nuclease resistance, an antisense agent can be further modified by inverting the nucleoside at the 3'-terminus with a 3'-3' linkage. In another alternative, the 3'- terminus can be blocked with an aminoalkyl group.

[00239] In other embodiments, an antisense oligonucleotide agent may include a modification that improves targeting, e.g., a targeting modification described herein.

[00240] Aptamers

[00241 ] Aptamers are nucleic acid molecules that bind a specific target molecule or molecules. Aptamers may be RNA or DNA based, and may include a riboswitch. A riboswitch is a part of an mRNA molecule that can directly bind a small target molecule, and whose binding of the target affects the gene's activity. Thus, an mRNA that contains a riboswitch is directly involved in regulating its own activity, depending on the presence or absence of its target molecule. [00242] An oligonucleotide agent featured in the invention can be an aptamer. An aptamer binds to a non-nucleic acid ligand, such as a small organic molecule or protein, e.g., a transcription or translation factor, and subsequently modifies (e.g., inhibits) activity. An aptamer can fold into a specific structure that directs the recognition of the targeted binding site on the non-nucleic acid ligand. An aptamer can contain any of the modifications described herein. [00243] Ribozymes are oligonucleotides having specific catalytic domains that possess endonuclease activity (Kim and Cech, Proc Natl Acad Sci U S A. 1987 Dec;84(24):8788-92; Forster and Symons, Cell. 1987 Apr 24;49(2):21 1-20). At least six basic varieties of naturally- occurring enzymatic RNAs are known presently. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[00244] Methods of producing a ribozyme targeted to any target sequence are known in the art. Ribozymes may be designed as described in PCT Publications WO 93/23569 and WO 94/02595, each specifically incorporated herein by reference, and synthesized to be tested in vitro and in vivo, as described therein.

[00245] Routes of Delivery

[00246] For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It may be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A composition that includes a iRNA can be delivered to a subject by a variety of routes. Exemplary routes include:

intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

[00247] Compositions comprising a polymer according to various embodiments of the invention may be provided in a unit dosage form, or in a bulk form suitable for formulation or dilution at the point of use. Such compositions may be administered to a subject in a single-dose, or in several doses administered over time. Dosage schedules may be dependent on, for example, the subject's condition, age, gender, weight, route of administration, formulation, or general health. Dosage schedules may be calculated from measurements of adsorption, distribution, metabolism, excretion and toxicity in a subject, or may be extrapolated from measurements on an experimental animal, such as a rat or mouse, for use in a human subject. Optimization of dosage and treatment regimens are discussed in, for example, Goodman & Gilman's The Pharmacological Basis of Therapeutics 1 1th edition. 2006. LL Brunton, editor. McGraw-Hill, New York, or Remington, The Science and Practice of Pharmacy, 21st edition. Gennaro et al. Editors. Lippincott Williams & Wilkins, Philadelphia. [00248] The amount of a composition administered, where it is administered, the method of administration and the timeframe over which it is administered may all contribute to the observed effect. As an example, a composition may be administered systemically e.g.

intravenous administration and have a toxic or undesirable effect, while the same composition administered subcutaneously may not yield the same undesirable effect. In some embodiments, localized stimulation of immune cells in the lymph nodes close to the site of subcutaneous injection may be advantageous, while a systemic immune stimulation may not.

[00249] Standard reference works setting forth the general principles of medical physiology and pharmacology known to those of skill in the art include: Fauci et al., Eds., Harrison 's Principles Of Internal Medicine, 14th Ed., McGraw-Hill Companies, Inc. (1998). [00250] The polymers according to various embodiments of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of nucleic acid, the polymer and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. [00251] The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration. [00252] The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the iRNA in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the iRNA and mechanically introducing the DNA.

[00253] Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. [00254] Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When compositions are required for oral use, the polymer composition can be combined with emulsifying and /or suspending agents. If desired, certain sweetening and/or flavoring agents can be added. . See, for example, Berge et al. (1977. J. Pharm Sci. 66:1-19), or Remington- The Science and Practice of Pharmacy, 21^st edition. Gennaro et al editors. Lippincott Williams & Wilkins Philadelphia (both of which are herein incorporated by reference).

[00255] Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

[00256] Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes may be controlled to render the preparation isotonic.

[00257] For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly( vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers.

[00258] Topical Delivery

[00259] For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified nucleic acids. It may be understood, however, that these formulations, compositions and methods can be practiced with other nucleic acids, e.g., modified nucleic acids, and such practice is within the invention. In some embodiments, an nucleic acid, e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a siRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, or precursor thereof) may be delivered to a subject via topical administration. "Topical administration" refers to the delivery to a subject by contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface. As mentioned above, the most common topical delivery is to the skin. The term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum. Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.

[00260] The term "skin," as used herein, refers to the epidermis and/or dermis of an animal. Mammalian skin consists of two major, distinct layers. The outer layer of the skin is called the epidermis. The epidermis is comprised of the stratum comeum, the stratum

granulosum, the stratum spinosum, and the stratum basale, with the stratum corneum being at the surface of the skin and the stratum basale being the deepest portion of the epidermis. The epidermis is between 50 μπι and 0.2 mm thick, depending on its location on the body.

[00261 ] Beneath the epidermis is the dermis, which is significantly thicker than the epidermis. The dermis is primarily composed of collagen in the form of fibrous bundles. The collagenous bundles provide support for, inter alia, blood vessels, lymph capillaries, glands, nerve endings and immunologically active cells.

[00262] One of the major functions of the skin as an organ is to regulate the entry of substances into the body. The principal permeability barrier of the skin is provided by the stratum corneum, which is formed from many layers of cells in various states of differentiation. The spaces between cells in the stratum corneum is filled with different lipids arranged in latticelike formations that provide seals to further enhance the skins permeability barrier.

[00263] The permeability barrier provided by the skin is such that it is largely

impermeable to molecules having molecular weight greater than about 750 Da. For larger molecules to cross the skin's permeability barrier, mechanisms other than normal osmosis must be used.

[00264] Several factors determine the permeability of the skin to administered agents. These factors include the characteristics of the treated skin, the characteristics of the delivery agent, interactions between both the drug and delivery agent and the drug and skin, the dosage of the drug applied, the form of treatment, and the post treatment regimen. To selectively target the epidermis and dermis, it is sometimes possible to formulate a composition that comprises one or more penetration enhancers that will enable penetration of the drug to a preselected stratum.

[00265] Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics. The dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle (inunction) or through the use of one or more penetration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy.

[00266] In addition, iontophoresis (transfer of ionic solutes through biological membranes under the influence of an electric field) (Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163), phonophoresis or sonophoresis (use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 166), and optimization of vehicle characteristics relative to dose position and retention at the site of administration (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 168) may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.

[00267] The compositions and methods provided may also be used to examine the function of various proteins and genes in vitro in cultured or preserved dermal tissues and in animals. The invention can be thus applied to examine the function of any gene. The methods of the invention can also be used therapeutically or prophylactically. For example, for the treatment of animals that are known or suspected to suffer from diseases such as psoriasis, lichen planus, toxic epidermal necrolysis, erythema multiforme, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lyme disease and viral, fungal and bacterial infections of the skin.

[00268] Pulmonary Delivery [00269] For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to nucleic acids. It may be understood, however, that these formulations, compositions and methods can be practiced with various nucleic acids , e.g., iRNA agents, modified iRNA agents, and such practice is within the invention. A composition that includes an iRNA agent, e.g., a double- stranded iRNA agent, or siRNA agent, {e.g., a precursor, e.g., a larger iRNA agent which can be processed into a siRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, or precursor thereof) can be administered to a subject by pulmonary delivery. Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, for example, iRNA, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.

[00270] Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellar and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are may be used. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self contained. Dry powder dispersion devices, for example, deliver drugs that may be readily formulated as dry powders. A iRNA composition may be stably stored as lyophilized or spray-dried powders by itself or in

combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.

[00271 ] The term "powder" means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli. Thus, the powder is said to be "respirable." For example, the average particle size is less than about 10 μπι in diameter with a relatively uniform spheroidal shape distribution. In some embodiments, the diameter is less than about 7.5 μιη and in some embodiments less than about 5.0 μηι. Usually the particle size distribution is between about 0.1 μηι and about 5 μηι in diameter, sometimes about 0.3 μηι to about 5 μιη.

[00272] The term "dry" means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and in some cases less it than about 3% w. A dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol. [00273] The term "therapeutically effective amount" is the amount of a therapeutic agent (e.g. nucleic acid) present in the composition that is needed to provide the desired level of therapeutic agent in the subject to be treated, to remedy, ameliorate or prevent a disease state, or give the anticipated physiological response. The therapeutic agent may increase or decrease the level of a gene product (e.g. a transcript or a polypeptide); the therapeutically effective amount of the agent may be determined with reference to, for example, the presence, absence or level of an enzymatic activity, apoptosis, metabolite, metabolic byproduct, waste product, polypeptide or the like.

[00274] The term "pharmaceutically acceptable carrier" means that the carrier can be taken into the lungs with no significant adverse toxicological effects on the lungs. [00275] The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two. [00276] Bulking agents that are particularly valuable include compatible carbohydrates, polypeptides, amino acids or combinations thereof. Suitable carbohydrates include

monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-beta-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like. A group of carbohydrates may includes lactose, trehalose, raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being used in some embodiments.

[00277] Additives, which are minor components of the composition of this invention, may be included for conformational stability during spray drying and for improving dispersibility of the powder. These additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, and the like.

[00278] Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate may be used in some embodiments. [00279] Pulmonary administration of a micellar iRNA formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane,

heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.

[00280] Oral or Nasal Delivery [00281 ] For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to nucleic acids. It may be understood, however, that these formulations, compositions and methods can be practiced with various nucleic acids, e.g., iRNA agents, modified iRNA agents, and such practice is within the invention. Both the oral and nasal membranes offer advantages over other routes of administration. For example, drugs administered through these membranes have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the drug to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the drug can be applied, localized and removed easily. [00282] In oral delivery, compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek. The sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many drugs. Further, the sublingual mucosa is convenient, acceptable and easily accessible. [00283] In certain other aspects, the invention provides kits that include a suitable container containing a pharmaceutical formulation comprising a nucleic acid, e.g., an iRNA agent, a double-stranded iRNA agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a siRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for an iRNA agent preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a

pharmaceutical composition. The kit can also include a delivery device.

[00284] In another aspect, the invention features a device, e.g., an implantable device, wherein the device can dispense or administer a composition that comprises a nucleic acid, e.g., an iRNA agents, a double-stranded iRNA agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a siRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, or precursor thereof), e.g., a iRNA agent that silences an endogenous transcript. In one embodiment, the device is coated with the composition. In another embodiment the iRNA agent is disposed within the device. In another embodiment, the device includes a mechanism to dispense a unit dose of the composition. In other embodiments the device releases the composition continuously, e.g., by diffusion. Exemplary devices include stents, catheters, pumps, artificial organs or organ components (e.g., artificial heart, a heart valve, etc.), and sutures.

[00285] Articles of Manufacture

[00286] Also provided is an article of manufacture, comprising packaging material and a composition comprising a polyacetal, polyketal or ether polymer as described herein, and a nucleic acid. The composition includes an excipient, and the packaging material may include a label which describes the components of the composition. The label may further include an intended use of the composition, for example as a therapeutic agent or a research tool to be used with kits as set out herein. [00287] Kits

[00288] A kit comprising a composition comprising a polyacetal, polyketal or ether polymer as described herein, along with instructions for use of the polymer or composition for delivery of a nucleic acid to a cell, or to an intracellular location within the cell. The kit may be useful for delivery of a nucleic acid (for example, an siRNA, antisense or iRNA) to a cell, or a tissue or cell of a subject to whom it is administered, and the instructions may include, for example, dose concentrations, dose intervals, preferred administration methods, methods for screening or testing, or assessing the effect of the nucleic acid on the subject, or the like.

[00289] In another embodiment, a kit for the preparation of a medicament, comprising a composition comprising a polyacetal, polyketal or ether polymer as described herein, along with instructions for its use is provided. The instructions may comprise a series of steps for the preparation of the medicament, the medicament being useful for delivery of a nucleic acid (for example, an siRNA, antisense or iRNA) to a cell, or to a tissue or cell of a subject to whom it is administered.

[00290] The present invention will be further illustrated in the following examples. However it is to be understood that these examples are for illustrative purposes only, and should not be used to limit the scope of the present invention in any manner.

SYNTHETIC METHODS The multifunction polymers of the invention can be prepared by the following synthetic schemes.

[00291] Example 1; Synthesis of monomer 2-{l-methyl-l-[2-(oxiran-2-yl methoxy) ethoxyl ethoxyi ethanol (Compound 5):

[00292] Figure 1 shows an exemplary synthetic scheme for the 2- { 1 -methyl- 1 -[2-(oxiran- 2-yl methoxy) ethoxy] ethoxy} ethanol monomer (Compound 5).

[00293] Ethylene glycol (1) (30 mL, 0.537 moles), trimethyl orthoacetate (90 mL, 0.698 moles) and p-toluene sulfonic acid (2 g) were dissolved in 600 mL of dichloromethane and stirred at room temperature for 4 h. After this, water (12.6 mL, 0.698 moles) was added and reaction mixture stirred for 1 h. Dichloromethane was removed under vacuum and the product 2- hydroxyethylacetate (2) was purified by flash chromatography on silica gel using chloroform: acetone (9: 1). Yield:70%

[00294] Compound 2 (22g, 0.211 moles) was dissolved in 300 mL THF. Pyridinium p- toluenesulfonate (PPTS, 2.2 g, 0.00844 moles) was added to it and stirred for 15 min. 5A molecular sieves (lOOg) was added to the mixture and stirred for additional 15 min. To this, 2- methoxypropene (8.4 mL, 0.0844 moles) was added and the reaction mixture was stirred at room temperature for 48 h. THF was removed under vacuum to yield propane-2,2-diylbis(oxyethane- 2,l-diyl)diacetate (3) as a pale yellow liquid. Yield: 60% [00295] Acetyl group deprotection of compound 3 was carried out by treating with sodium hydroxide in methanol/water at room temperature. After the reaction, brine was added and the product extracted with dichloromethane. Upon removal of dichloromethane under vacuum, 2,2'- [propane-2,2-diylbis(oxy)] diethanol (4) was obtained as yellow viscous oil. Yield: 40%

[00296] Sodium (0.85 g, 0.0366 moles) and isopropanol (80 mL) was refluxed for 4 h. The solution was cooled to room temperature and the compound 4 (6 g, 0.0366 moles) was added slowly with stirring. To this reaction mixture, epichlorohydrin (28 mL, 0.366 moles) was added dropwise and stirred at room temperature for 15 h. The salt NaCl was filtered off and

isopropanol was removed under vacuum. The compound was dissolved in dichloromethane and washed with water. Dichloromethane was removed under vacuum to yield the compound 5 as yellow oil. The product was further purified by column chromatography on silica gel containing 1 % triethylamine using hexane/ethyl acetate as the eluent. Yield : 60%

[00297] In the last step of the synthetic scheme, a mixture of mono and diepoxide is formed. The Ή NMR spectra of the crude monomer is shown in Figure 2. The monoepoxide and diepoxide were separated by column chromatography on silica gel neutralized with triethylamine. A mixture of hexane and ethyl acetate containing 1% triethylamine was used as the eluent. The ^!H NMR spectra of the monoepoxide (ketal monomer) is shown in Figure 3, while the Ή NMR spectra of the diepoxide is shown in Figure 4. Mass spectroscopic analysis demonstrated a peak at 243 (M-Na⁺) whereas the diepoxide exhibits peak at 299 (M- Na⁺). The monoepoxide shows a peak at 2.4 ppm in the NMR spectrum corresponding to the hydroxyl proton, which is absent in the spectrum of the diepoxide. The presence of the diepoxide monomer may result in crosslinking during the later polymerization reactions, and its removal may be warranted for some applications. For the subsequent polymerizations described herein, the diepoxide was removed before polymerization. For all subsequent syntheses (unless otherwise specified), chromatographically purified ketal monomer was used.

[00298] 1H NMR (CDC1₃, 400 MHz): δ ppm 1.38 ( 6 H), 2.3( 1H), 2.62 (1 H), 2.79 (1 H), 3.16 (1 H), 3.40 (1 H), 3.60-3.64(6 H), 3.72 (2 H), 3.82 (1 H)

[00299] ^I3C NMR (CDC1₃, 400 MHz) : δ ppm 24.72, 44.01, 50.66, 60.05,

[00300] Example 2: Synthesis of poly(ketal hydroxyalkane)(PKHA) homopolymer by polymerization of compound 5

[00301] Ketal monomer 5 was initially tested for polymerization behavior using 1,1,1- Tris(hydroxymethyl)propane (TMP) and potassium methylate as the initiator. Figure 6 shows an exemplary, general synthetic scheme for the synthesis of the homopolymer by polymerization of Compound 5. In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.050 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 95 °C and the ketal monomer 5 (2 g, 0.009 moles) was added over a period of 6 h using a syringe pump. After complete addition of monomer, the reaction mixture was stirred for an additional 2 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum. Number average molecular weight (Mn) was determined by gel-permeation chromatography (GPC). A ratio of Mw/Mn provides a polydispersity index (PDI).

[00302] Mn (from GPC): 13000 Da; Mw/Mn: 1.7

[00303] NMR spectrum of the polymer (Figure 5) shows peak at 1.36 ppm characteristic of ketal methyl protons and peaks in the region 3.5-4.0 ppm due to the other methylene protons in the chain. A GPC trace of the polymer (in 0.1 M NaN0₃ at pH 8.5) exhibited a monomodal molecular weight distribution with a molecular weight of 13000 and polydispersity of 1.7.

[00304] A pH dependent degradation kinetics study was performed on this homopolymer. 12-15 mg of polymer was dissolved in 0.6 mL of buffers of different pH (in D₂0) and their NMR spectra were recorded with respect to time. Percentage of degradation was calculated from the ratio of the integrals of the peak at 2.18 ppm (due to acetone) and 1.37 ppm (due to ketal group). Results are summarized in Table 5.

Table 5. pH dependent degradation of 13 KDa/PDI 1.7 PKHA homopolymer

[00305] Example 3: Synthesis of poly(ketal hydroxyalkanes)(PKHA) homopolymer by polymerization of compound 5 [00306] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.050 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 75°C and the ketal monomer 5 (1 g, 0.0045 moles) was added over a period of 6 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 2 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum.

[00307] Ή NMR (D₂0, 300 MHz): δ ppm 1.37 (ketal CH₃ protons), 3.2-4.0 (methylene and methine protons from ketal monomer) [00308] Mn (from GPC): 6000 Da

[00309] Example 4: Synthesis of poly(ketal hydroxyalkanes)(PKHA) homopolymer by polymerization of compound 5

[00310] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.050 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 95°C and the ketal monomer 5 (6 g, 0.027 moles) was added over a period of 24 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 24 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum. [0031 1] Ή NMR (D₂0, 300 MHz): 6 ppm 1.37 (ketal CH₃ protons), 3.2-4.0 (methylene and methine protons from ketal monomer)

[00312] Mn (from GPC): 45000 Da

[00313] Mw/Mn : 1.22

[00314] Example 5: Initial synthesis of PKHA copolymer by copolymerization of compound 5 and glycidol (with crude monomer) and pH sensitivity of polymer

[00315] Ketal monomer (before chromatographic separation from the di epoxide) and glycidol were tested for copolymerization using a TMP/potassium methylate initiator. A mixture of crosslinked and methanol-soluble polymers was obtained in all 3 tests, as was observed in monomer polymerization experiments; cross-linking may be due to the traces of diepoxide in the crude monomer. For all subsequent syntheses, chromatographically purified ketal monomer was used.

[00316] Figure 8 shows an exemplary general scheme for synthesis of the copolymer by polymerization of compound 5 with glycidol.

[00317] Molecular weight of the polymers determined by GPC in 0.1 M NaN0₃ exhibited lower valued and higher polydispersity generally. Without wishing to be bound by theory, this may be due to partial degradation of the polymer in the NaN0₃ diluent, which undergoes acification over time. To address this, GPC of samples were also run using 0.1 M NaN0₃ buffered to pH 8.5 (phosphate buffer). The resulting chromatogram demonstrated a lower polydispersity, and a higher Mn, as illustrated in Figure 7a, b. Based on these results, GPC analysis was conducted at a basic pH for all polymers.

[00318] Example 6: Synthesis of PKHA copolymer by co-polymerization of compound 5 and glycidol [00319] Table 6 shows the results of the copolymerization of ketal monomer (after purification by column chromatography) and glycidol. As is evident from the table, higher monomer conversions and better control over the molecular weight and polydispersity was achieved. Polymerizations were successful both at 70 and 95°C which shows the robustness of this system. Reactivity of the ketal monomer appears to be lower compared to glycidol resulting in polymers with lower incorporation of this monomer. Monomodal peak was observed in the GPC traces of these polymers., while polymers prepared using unpurified monomers

demonstrated two or more peaks.

[00320] Exemplary synthetic methods are provided in Examples 6a to 6f.

Table 6. Colymerization of ketal monomer (following diepoxide separation) and glycidol

2 0.30 0.030 95 12 80 42 58 2300 1.9

3 2.0 0.20 95 16 75 56 44 8500 1.4

[00321] Example 6a: Copolymer synthesis (70°C. 3:1 molar ratio of monomer: glycidol)

[00322] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.050 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 70°C and a mixture of the ketal monomer 5 (2 g, 0.0090 moles) and glycidol (0.2 mL, 0.003 moles) was added over a period of 12 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 4 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum.

[00323] Mn (from GPC): 6600 Da [00324] Mw/Mn : 1.7

[00325] Physical characteristics: The polymer was soluble in water as well as in organic solvents such as chloroform, methanol, tetrahydrofuran and N,N-dimethyl formamide. Polymer composition was calculated from NMR from the integrals of the peak at 1.37 ppm ( due to ketal group) and peaks in the region 3.5-4.0 ppm.

[00326] Example 6b: Copolymer synthesis (95°C, 3: 1 molar ratio of monomer: glycidol)

[00327] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.050 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 95°C and a mixture of the ketal monomer 5 (2 g, 0.0090 moles) and glycidol (0.2 mL, 0.003 moles) was added over a period of 12 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 4 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum.

[00328] Mn (from GPC): 8500 Da [00329] Mw/Mn : 1.4

[00330] Ή NMR spectrum of the resulting copolymer is shown in Figure 9, and a GPC trace of the resulting copolymer is shown in Figure 10.

[00331] Example 6c: Copolymer synthesis (95°C, 3:1 molar ratio of monomer: glvcidol) [00332] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.050 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 95°C and a mixture of the ketal monomer 5 (5 g, 0.0225 moles) and glycidol (0.5 mL, 0.0075 moles) was added over a period of 12 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 4 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum.

[00333] Mn (from GPC): 48000 Da

[00334] Example 6d: Copolymer synthesis (70°C, 3:1 molar ratio of monomer: glvcidol) [00335] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.050 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 70°C and a mixture of glycidol (0.2 mL, 0.003 moles, 25 mol%) and the ketal monomer 5 (2 g, 0.009 moles, 75 mol%) was added over a period of 6 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 2 h. The polymer was dissolved in methanol and precipitated twice from diethyl ether. The polymer was dried under vacuum.

[00336] Physical characteristics: The polymer was soluble in water as well as in organic solvents such as chloroform, methanol, tetrahydrofuran and Ν,Ν-dimethyl formamide. [00337] Ή NMR (D₂0, 300 MHz): δ ppm 1.37 (ketal CH₃ protons), 3.2-4.0 (CH₂ and CH protons from glycidol and ketal monomer) (Figure 11). [00338] Composition calculated from NMR was found to be 60 mol% ketal monomer and 40 mol% glycidol.

[00339] Mn (from GPC): 13000 Da

[00340] Mw/Mn : 1.60 [00341] Example 6e: Copolymer synthesis (70°C, 3: 1 molar ratio of monomer: glycidol)

[00342] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.050 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 70°C and a mixture of glycidol (0.1 mL, 0.0015 moles, 25 mol%) and the ketal monomer 5(1 g, 0.0045 moles, 75 mol%) was added over a period of 6 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 2 h. The polymer was dissolved in methanol and precipitated twice from diethyl ether. The polymer was dried under vacuum. The polymer was soluble in water as well as in organic solvents such as chloroform, methanol and acetone.

[00343] 1H NMR (D₂0, 300 MHz): δ ppm 1.37 (ketal CH₃ protons), 3.2-4.0 (CH₂ and CH protons from glycidol and ketal monomer)

[00344] Composition calculated from NMR was found to be 40 mol% glycidol and 60 mol% ketal monomer [00345] Mn (from GPC): 7000 Da

[00346] Mw/Mn : 2.0

[00347] Example 6f: Copolymer synthesis (95°C. 3:1 molar ratio of monomer: glycidol)

[00348] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.090 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 95 °C and a mixture of the ketal monomer 5 (7.2 g, 0.0327 moles) and glycidol (0.7 mL, 0.0108 moles) was added over a period of 20 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 10 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum. Yield: 4g.

[00349] Mn : 22000 Da [00350] Mw/Mn : 1.4

[00351 ] Composition of the copolymer calculated from NMR was found to be 60% ketal monomer and 40% glycidol

[00352] A GPC trace of the polymer is shown in Figure 12.

[00353] Example 6g: Copolymer synthesis (95°C, 1 :9 molar ratio of monomer: glycidol) [00354] Synthesis of high molecular weight copolymer was done, using 1 ,4-dioxane as the emulsifying agent. Molecular weight of the polymer determined by GPC was found to be 480000 Da.

[00355] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.090 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. 1,4-Dioxane (3.6 mL) was added and the flask was heated to 95°C. A mixture of the ketal monomer 5 (2.3 g, 0.0104 moles) and glycidol (6.3 mL, 0.0945 moles) was added over a period of 16 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 4 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum.

[00356] Mn : 480000 Da [00357] Mw/Mn : l .l

[00358] Composition of the copolymer calculated from NMR was found to be 7 mol% ketal monomer and 93 mol% glycidol (Figure 13). Molecular weight of the copolymer measured by GPC after acidolysis with 0.1 M HCl was found to be 300000 Da with a polydispersity of 1.2. However in the NMR spectrum, the peak due to ketal group disappeared completely after degradation. This result suggests that the ketal comonomer may not be incorporated uniformly in the copolymer; instead the majority may be present on the solvent-exposed surface of the polymer.

[00359] Without wishing to be bound by theory, a more uniform incorporation of the ketal comonomer may be obtained, for example, by synthesizing a core of the ketal monomer (e.g. an initial homopolymer), followed by extension of the chain with glycidol.

[00360] Example 7. Degradation kinetics of poly(ketal hydroxyalkanes) polymers at different pHs

[00361] The kinetics of degradation of the various polymers was studied by dissolving the polymer in different buffers in D₂0 and monitoring the disappearance of peak at 1.37 ppm (due to ketal group) and appearance of a new peak at 2.18 ppm (due to the formation of acetone) in NMR. Aqueous solutions of poly (ketal hydroxyalkanes) polymer were prepared in different buffers in D₂0 and the degradation was analyzed.

[00362] All the polymers behaved similarly to the change in pHs irrespective of molecular weight, composition and polydispersity. [00363] Figure 14 illustrates the kinetics of degradation of a poly(ketal hydroxyalkane) copolymer as measured from 1H NMR peak integration at varying pH over time. The polymer was synthesized according to Example 6d (Mn 13000 Da by GPC, Mw/Mn: 1.6).

[00364] Figure 15 shows Ή MNR spectra of the polymer of example 6d as it is incubated at pH 5.5 for 0-2 hours. Figure 16 shows 1H MNR spectra of the polymer of example 6d as it is incubated at pH 5.5 from 0.5 to 72 hours.

[00365] Figure 17 shows a pH dependent degradation profile of the PKHA homopolymer described in Example 4 (Mn 45000 Da; Mw/Mn: 1.22). Table 7 sets out the % degradation at the various pH over time for the PKHA homopolymer of Example 4.

Table 7. pH dependent degradation of 45 KDa PKHA homopolymer

Time (h) % Degradation at pH

1.1 5.5 6.4

1 100 78 17

1.5 94 28

2 100 38

2.5 47

3 54

[00366] Figure 18 shows a degradation profile at pH 6.4 of the PKHA homopolymers described in Example 2 (Mn 13000 Da; Mw/Mn: 1.7 )_ and Example 4 (Mn 45000 Da; Mw/Mn: 1.22) [00367] Figure 19 shows a pH dependent degradation profile of the PKHA copolymer described in Example 6f (Mn 22000 Da; Mw/Mn: 1.4). Table 8 sets out the % degradation at the various pH over time for the PKHA copolymer of Example 6f.

Table 8. pH dependent degradation of 22 KDa PKHA copolymer

[00368] Figure 20 shows a degradation profile at pH 6.0 of the PKHA copolymers described in Example 6b (Mn 8500 Da; Mw/Mn: 1.4)_ and Example 6f (Mn 22000 Da; Mw/Mn: 1.4) Table 9 sets out the % degradation at the various pH over time for the PKHA copolymer of Example 6d.

Table 9. pH dependent degradation of 8.5 KDa PKHA copolymer

Time (h) % Degradation at pH

1.1 4.0 5.5 6.0 6.4 7.4

0.5 100 100 65 29 13

1 88 51 27 3.2

1.5 95 69 39

2 100 79 49 6.5

2.5 86 58 91 66 10

6 98.5 87 18

8 100 93

10 97 28

12 100

24 55

48 79

72 88

96 93

120 97

144 100

Table 10 sets out the % degradation at the various pH over time for the PKHA copolymer of Example 6g.

Table 10. pH dependent degradation of 480 KDa PKHAcopolymer

[00369] Polymers were tested for stability at a concentration of 12- 15mg/0.6 mL, using buffers at the indicated pH. This concentration was maintained for all polymers tested. For buffers of pH 5.5 and greater, phosphate buffer was used; acetate buffer for pH 4.0, and hydrochloric acid/KCl for pH 1.1.

[00370] Example 8. General protocol for the synthesis of amine-functionalized PKHA homo and copolymers

[00371] Amine functionalization of the polymer was carried out using N-(2,3-epoxypropyl phthalimide) followed by hydrazinolysis (Figure 21).

[00372] PKHA copolymer 13 kDa (0.08 g) was dried under vaccum for 48 h. The dried polymer was dissolved in 5 mL of anhydrous dimethyl formamaide. Potassium hydride (0.003 g) was taken in a round bottomed flask and washed with hexane. The polymer solution was added to the flask and stirred at RT for 2 h. N-(2,3-epoxypropyl phthalimide) (0.0063 g) dissolved in DMF was added and the reaction mixture was stirred at room temperature for 24 h. The polymer was precipitated twice from diethyl ether and dried under vaccum. The polymer was dissolved in methanol and refluxed with excess of hydrazine monohydrate for 48 h. The polymer was precipitated from diethyl ether. Figure 22 shows the NMR spectrum of the phthalimide functionalized 13 kDa copolymer. The amine-functionalized polymer was further reacted with NHS ester of fluorescein carboxylic acid. After work up, the polymer exhibited a reddish brown colour which indicates the successful conjugation of the fluorophore to the polymer.

[00373] Example 9. General protocol for the synthesis of azide-functionalized PKHA homo and copolymers

[00374] Dried PKHA polymer was prepared as per Example 8, and dissolved in anhydrous dichloromethane. To this, N₃-(PEG)₄-COOH, dicyclohexyl carbodiimide and 4-dimethylamino pyridine were added and the reaction mixture was stirred at room temperature for 48 h. The solution was centrifuged to removed the precipitated diurea and dichloromethane was removed under vacuum. The polymer was dissolved in methanol and precipitated from diethyl ether. Figure 23 shows the NMR spectrum of azide functionalized 6.6 kDa PKHA copolymer. [00375] Example 10a: Synthesis of other ketal monomers, polymerization and modification:

81

[00376] Example 10b: Chemical modification of the poly(ketal hydroxyalkane) copolymers:

• Carboxyl, amine and azido modification of the polymers

• Conjugation with cell surface targeting ligands

• Conjugation with siRNA

[00377] Example 11: Oligonucleotide Synthesis and Purification

[00378] Step 1. Oligonucleotide Synthesis

[00379] All oligonucleotides were synthesized on an AKTAoligopilot synthesizer or on an ABI 394 DNA/RNA synthesizer. Commercially available controlled pore glass solid supports (rU-CPG, 2'-0-methly modified rA-CPG and 2'-O-methyl modified rG-CPG from Prime Synthesis) or the in-house synthesized solid support hydroxyprolinol-cholesterol-CPG were used for the synthesis. RNA phosphoramidites and 2'-0-methyl modified RNA phosphoramidites with standard protecting groups (5'-0-dimethoxytrityl-N6-benzoyl-2'-t-butyldimethylsilyl- adenosine-3'-0-N,N'-diisopropyl-2-cyanoethylphosphoramidite, 5'-0-dimethoxytrityl-N4- acetyl-2'-i-butyldimethylsilyl-cytidine-3 '-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, 5'- 0-dimethoxytrityl-N2-isobutryl-2 ' -t-butyldimethylsilyl-guanosine-3 ' -Ο-Ν,Ν' -diisopropyl-2- cyanoethylphosphoramidite, 5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-0-N,N'- diisopropyl-2-cyanoethylphosphoramidite, 5'-O-dimethoxytrityl-N6-benzoyl-2'-O-methyl- adenosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, 5'-0-dimethoxytrityl-N4- acetyl-2'-O-methyl-cytidine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, 5'-O- dimethoxytrityl-N2-isobutryl-2 ' -O-methyl-guanosine-3 ' -O-N,N ' -diisopropyl-2- cyanoethylphosphoramidite, 5 ' -O-dimethoxytrityl-2 ' -O-mefhyl-uridine-3 ' -Ο-Ν,Ν' -diisopropyl-2- cyanoethylphosphoramidite and 5 ' -O-dimethoxytrityl-2 ' -deoxy-thymidine-3 ' -Ο-Ν,Ν ' - diisopropyl-2-cyanoethylphosphoramidite) were obtained from Pierce Nucleic Acids

Technologies and ChemGenes Research. The Quasar 570 phosphoramidite was obtained from Biosearch Technologies. The 5'-O-dimethoxytrityl- 2'-t-butyldimethylsilyl-inosine-3'-O-N,N'- diisopropyl-2-cyanoethylphosphoramidite was obtained from ChemGenes Research. The 5'-O- dimethoxytrityl- 2 ' -t-butyldimethylsilyl-(2,4)-diflurotolyl-3 '-Ο-Ν,Ν ' -diisopropyl-2- cyanoethylphosphoramidite (DFT-phosphoramidite) and the 5'-O-dimethoxytrityl- 2'-t- butyldimethylsilyl-9-(2-aminoethoxy)-phenoxazine-3'-O-N,N'-diisopropyl-2- cyanoethylphosphoramidite (G-clamp phosphoramidite) were synthesized in house.

[00380] For the syntheses on AKTAoligopilot synthesizer, all phosphoramidites were used at a concentration of 0.2 M in CH₃CN except for guanosine and 2'-0-methyl-uridine, which were used at 0.2 M concentration in 10% THF/CH₃CN (v/v). A coupling/recycling time of 16 minutes was used for all phosphoramidite couplings. The activator was 5-ethyl-thio-tetrazole (0.75 M, American International Chemicals). For the PO-oxidation, 50 mM iodine in water/pyridine

(10:90 v/v) was used and for the PS-oxidation 2% PADS (GL Synthesis) in 2,6-lutidine/CH₃CN (1 :1 v/v) was used. For the syntheses on ABI 394 DNA/RNA synthesizer, all phosphoramidites, including DFT and G-clamp phosphoramidites were used at a concentration of 0.15 M in CH₃CN except for 2 '-O-methyl -uridine, which was used at 0.15 M concentration in 10% THF/CH₃CN (v/v). A coupling time of 10 minutes was used for all phosphoramidite couplings. The activator was 5-ethyl-thio-tetrazole (0.25 M, Glen Research). For the PO-oxidation, 20 mM iodine in water/pyridine (Glen Research) was used and for the PS-oxidation 0.1M DDTT (AM Chemicals) in pyridine was used. Coupling of the Quasar 570 phosphoramidite was carried out on the ABI DNA/RNA synthesizer. The Quasar 570 phosphoramidite was used at a concentration of 0.1 M in CH₃CN with a coupling time of 10 mins. The activator was 5-ethyl-thio-tetrazole (0.25 M, Glen Research) and 0.1M DDTT (AM Chemicals) in pyridine was used for PS oxidation.

[00381 ] Step 2. Deprotection of oligonucleotides

[00382] A. Sequences synthesized on the AKTAoligopilot synthesizer

[00383] After completion of synthesis, the support was transferred to a 100 mL glass bottle (VWR). The oligonucleotide was cleaved from the support with simultaneous deprotection of base and phosphate groups with 40 mL of a 40% aq. methyl amine (Aldrich) 90 mins at 45°C. The bottle was cooled briefly on ice and then the methylamine was filtered into a new 500 mL bottle. The CPG was washed three times with 40 mL portions of DMSO. The mixture was then cooled on dry ice.

[00384] In order to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2' position, 60 mL tnethylamine trihydrofluoride (Et3N-HF) was added to the above mixture. The mixture was heated at 40°C for 60 minutes. The reaction was then quenched with 220 mL of 50 mM sodium acetate (pH 5.5) and stored in the freezer until purification.

[00385] B. Sequences synthesized on the ABI DAN/RNA synthesizer

[00386] After completion of synthesis, the support was transferred to a 15 mL tube (VWR). The oligonucleotide was cleaved from the support with simultaneous deprotection of base and phosphate groups with 7 mL of a 40% aq. methyl amine (Aldrich) 15 mins at 65°C. The bottle was cooled briefly on ice and then the methylamine was filtered into a 100 mL bottle (VWR). The CPG was washed three times with 7 mL portions of DMSO. The mixture was then cooled on dry ice. [00387] In order to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2' position, 10.5 mL triethylamine trihydrofluoride (Et3N-HF) was added to the above mixture. The mixture was heated at 60°C for 15 minutes. The reaction was then quenched with 38.5 mL of 50 mM sodium acetate (pH 5.5) and stored in the freezer until purification.

[00388] Step 3. Quantitation o Crude Oligonucleotides [00389] For all samples, a 10 aliquot was diluted with 990 of deionised nuclease free water (1.0 mL) and the absorbance reading at 260 nm was obtained.

[00390] Step 4. Purification of Oligonucleotides

[00391] (a) Unconjugated oligonucleotides

[00392] The unconjugated crude oligonucleotides were first analyzed by HPLC (Dionex PA 100). The buffers were 20 mM phosphate, pH 11 (buffer A); and 20 mM phosphate, 1.8 M NaBr, pH 1 1 (buffer B). The flow rate 1.0 mL/min and monitored wavelength was 260-280 nm. Injections of 5-15 μL were done for each sample. [00393] The unconjugated samples were purified by HPLC on a TSK-Gel SuperQ-5PW (20) column packed in house (17.3 x 5 cm) or on a commercially available TSK-Gel SuperQ- 5PW column (15 x 0.215cm) available from TOSOH Bioscience. The buffers were 20 mM phosphate in 10% CH₃CN, pH 8.5 (buffer A) and 20 mM phosphate, 1.0 M NaBr in 10%

CH₃CN, pH 8.5 (buffer B). The flow rate was 50.0 mL/min for the in house packed column and lO.Oml/min for the commercially obtained column. Wavelengths of 260 and 294 nm were monitored. The fractions containing the full-length oligonucleotides were pooled together, evaporated, and reconstituted to -100 mL with deionised water.

[00394] (b) Cholesterol-conjugated oligonucleotides [00395] The cholesterol-conjugated crude oligonucleotides were first analyzed by LC/MS to determine purity. The cholesterol conjugated sequences were HPLC purified on RPC- Sourcel5 reverse-phase columns packed in house (17.3 x 5 cm or 15 x 2 cm). The buffers were 20 mM NaOAc in 10 % CH₃CN (buffer A) and 20 mM NaOAc in 70% CH₃CN (buffer B). The flow rate was 50.0 mL/min for the 17.3x 5cm column and 12.0 mL/min for the 15 x 2 cm column. Wavelengths of 260 and 284 nm were monitored. The fractions containing the full- length oligonucleotides were pooled, evaporated, and reconstituted to 100 mL with deionised water.

[00396] Step 5. Desalting of Purified Oligonucleotides

[00397] The purified oligonucleotides were desalted on either an AKTA Explorer or an AKTA Prime system (Amersham Biosciences) using a Sephadex G-25 column packed in house. First, the column was washed with water at a flow rate of 40 mL/min for 20-30 min. The sample was then applied in 40-60 mL fractions. The eluted salt-free fractions were combined, dried, and reconstituted in -50 mL of RNase free water.

[00398] Step 6. Purity Analysis by Capillary Gel Electrophoresis (CGE), Ion-exchange HPLC (IEX), and Electrospray LC/Ms

[00399] Approximately 0.3 OD of each of the desalted oligonucleotides were diluted in water to 300 and were analyzed by CGE, ion exchange HPLC, and LC/MS.

[00400] Step 7. Duplex formation [00401] For the fully double stranded duplexes, equimolar amounts of two strands were mixed together. The mixtures were frozen at -80°C and dried under vacuum on a speed vac. Dried samples were then dissolved in l PBS to a final concentration of 40 mg/mL. The dissolved samples were heated to 95°C for 5 min and slowly cooled to room temperature. [00402] Step 8. Tm determination

[00403] For the partial double stranded duplexes and hairpins melting temperatures were determined. For the duplexes, equimolar amounts of the two single stranded RNAs were mixed together. The mixtures were frozen at -80°C and dried under vacuum on a speed vac. Dried samples were then dissolved in lxPBS to a final concentration of 2.5μΜ. The dissolved samples were heated to 95°C for 5 min and slowly cooled to room temperature. Denaturation curves were acquired between 10 - 90 °C at 260 nm with temperature ramp of 0.5 °C/min using a Beckman spectrophotometer fitted with a 6-sample thermostated cell block. The Tm was then determined using the 1st derivative method of the manufacturer's supplied program.

[00404] Example 12: 2 -F Oligonucleotide Synthesis [00405] 1. Oligonucleotide Synthesis:

[00406] All oligonucleotides were synthesized on an AKTAoligopilot synthesizer.

Commercially available controlled pore glass solid support (dT-CPG, 500 , Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5'-0-dimethoxytrityl N6-benzoyl- 2 ' -t-butyldimethylsilyl-adenosine-3 ' - ΟΝ,Ν ' -diisopropyl-2-cyanoethylphosphoramidite, 5 ' -O- dimethoxytrityl-N4-acetyl-2 ' -t-butyldimethylsilyl-cytidine-3 ' -Ο-Ν,Ν' -diisopropyl-2- cyanoethylphosphoramidite, 5 '-0-dimethoxytrityl-N2~isobutryl-2 '-t-butyldimethylsilyl- guanosine-3 ' -Ο-Ν,Ν' -diisopropyl-2-cyanoethylphosphoramidite, and 5 ' -O-dimethoxytrityl-2 ' -t- butyldimethylsilyl-uridine-3 '-0-N,N'-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis. The 2'-F phosphoramidites, 5'-0-dimethoxytrityl-N4-acetyl-2'-fluro-cytidine-3'-0-N,N'-diisopropyl-2-cyanoethyl- phosphoramidite and 5 ' -O-dimethoxytrityl-2 ' -fluro-uridine-3 '-Ο-Ν,Ν' -diisopropyl-2-cyanoethyl- phosphoramidite were purchased from (Promega). All phosphoramidites were used at a concentration of 0.2M in acetonitrile (CH₃CN) except for guanosine which was used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes was used. The activator was 5-ethyl thiotetrazole (0.75M, American International Chemicals), for the PO- oxidation Iodine/Water/Pyridine was used and the PS-oxidation PADS (2 %) in 2,6- lutidine/ACN (1 :1 v/v) was used. The cholesterol phosphoramidite was synthesized in house, and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite was 16 minutes. [00407] Deprotection- 1 (Nucleobase Deprotection)

[00408] After completion of synthesis, the support was transferred to a 100 mL glass bottle(VWR). The oligonucleotide was cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6.5h at 55°C. The bottle was cooled briefly on ice and then the ethanolic ammonia mixture was filtered into a new 250 mL bottle. The CPG was washed with 2 x 40 mL portions of ethanol/water (1 :1 v/v). The volume of the mixture was then reduced to ~ 30 mL by roto-vap. The mixture was then frozen on dyince and dried under vacuum on a speed vac.

[00409] 3. Deprotection-II (Removal of 2 ' TBDMS group)

[00410] The dried residue was resuspended in 26 mL of triethylamine,

triethylamine trihydrofluoride (TEA.3HF) and DMSO (3 :4:6) and heated at 60°C for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2' position. The reaction was then quenched with 50 mL of 20 mM sodium acetate and pH adjusted to 6.5, and stored in freezer until purification.

[0041 1] 4. Quantitation of Crude Oligomer or Raw Analysis [00412] For all samples, a 10 μΐ aliquot was diluted with 990 μΐ of deionised nuclease free water (1.0 mL) and absorbance reading obtained at 260 nm.

[00413] Purification of Oligomers

[00414] (a) HPLC Purification

[00415] The crude oligomers were first analyzed by HPLC (Dionex PA 100). The buffer system was: A = 20 mM phosphate pH 11 , B = 20 mM phosphate, 1.8 M NaBr, pH 11 , flow rate 1.0 mL/min, and wavelength 260-280 nm. Inject 5-15 μΐ of the each sample. The unconjugated samples were purified by HPLC on a TSK-Gel SuperQ-5PW (20) column packed in house (17.3 x 5 cm). The buffer system was: A = 20 mM phosphate in 10% ACN, pH 8.5 and B = 20 mM phosphate, 1.0 M NaBr in 10% ACN, pH 8.5, with a flow rate of 50.0 mL/min, and wavelength 260 and 294. The 5 '-cholesterol conjugated sequences were HPLC purified using a reverse-phase column. The buffer system was: A = 20 mM TEAA in 10 % ACN and B = 20 raM TEAA in 70% ACN. The fractions containing the full length oligonucleotides were then pooled together, evaporated and reconstituted to 100 mL with deionised water.

[00416] 6. Desalting of Purified Oligomer

[00417] The purified oligonucleotides were desalted using AKTA Explorer (Amersham Biosciences) using Sephadex G-25 column. First column was washed with water at a flow rate of 25 mL/min for 20-30 min. The sample was then applied in 25 mL fractions. The eluted salt-free fractions were combined together, dried down and reconstituted in 50 mL of RNase free water.

[00418] 7. Capillary Gel Electrophoresis (CGE) and Electrospray LC/MS

[00419] Approximately 0.15 OD of desalted oligonucleotides were diluted in water to 150 μΐ and then pipetted into vials for CGE and LC/MS analysis.

[00420] Example 13. Synthesis of PKHA copolymer by copolymerization of compound 5 and glycidol

[00421] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.040 g). To this potassium methylate (25% solution in methanol, 0.075 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 95°C and a mixture of the ketal monomer 5 (6.5 g, 0.0295 moles) and glycidol (0.65 mL, 0.0098moles) was added over a period of 20 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 10 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum.

[00422] Mn (GPC in chloroform) : 28 200 [00423] Mw/Mn : 1.44

[00424] Composition of the copolymer calculated from NMR was found to be 66% ketal monomer and 34% glycidol (Figure 24). Example 14. Synthesis of 2-(l-(2-(oxiran-2-ylmethoxy) ethoxy) cyclohexyloxy)

[00426] To a mixture of cyclohexanone (6) (lOOg) and trimethyl orthoformate (130 g), was added p-Toluene sulfonic acid (1.9 g) in portions at RT and stirred for lhr. It was then connected to fractional distillation unit with long vigreux column and distilled the mixture at atmospheric pressure. Methyl formate and methanol were removed first then trimethyl ortho formate. The fraction distilled at 125-138°C was collected as pure compound 7. Yield: 105 g (92%). [00427] To a solution of ethylene glycol monoacetate (104.4 g) in THF (1.5 L), was added p-toluenesulfonic acid (9.53 g) and molecular sieves (5 A) (475 g). The mixture was stirred for 15 min. Compound 7 (45 g) was added as one portion and stirred at room temperature for 48hours (TLC). Then 9.5g of sodium bicarbonate was added to the reaction mass and stirred for lOmin. It was filtered through a pad of K₂C0₃ kept over celite bed. The filtrate was evaporated to obtain crude compound 8, which was purified by silica gel chromatography using 2-5% ethyl acetate in hexane as eluent. Yield: 112g (46%)

[00428] To a stirred solution of compound 8 (1 12 g) in methanol (1.1 L), was added potassium carbonate(161.6 g) and water (56 mL). Stirring was continued at RT for 15hours (TLC). Reaction mixture was filtered through celite bed, washed with methanol and evaporated the filtrate. The residue obtained was triturated with excess diethyl ether (5 x 2L), and evaporated the ethereal solution. The product obtained was triturated again with hexane (2 x 100ml) and decanted the hexane layer. The viscous product was then dissolved in diethylether, dried over sodium sulfate, filtered and evaporated to afford pure compound 9. Yield: 75 g (94%) [00429] To a solution of potassium-t-butoxide (2.75 g) in t-butanol (75ml) at RT under nitrogen, was added a solution of compound 8 (5 g) in t-butanol (50ml) slowly with stirring during 15min. Stirring was continued for additional 15min. Then ephichlorohydrin (18.2 g) was added drop wise over a period of 20min and stirred at room temperature for 15hours.The salt formed was filtered off and the filtrate was evaporated at reduced pressure to yield a mixture of product and unreacated diol. The crude product was dissolved in 50ml of dichloromethane and washed with water (lx 10ml). The layers were separated and the organic layer was evaporated at reduced pressure. The compound 10 was further purified by flash chromatography through silica gel neutralized with triethylamine. Product was eluted at 2-20% ethyl acetate/hexane containing 1% triethylamine as the eluent. Yield: 2.6 g (40%)

[00430] H MR-CDCI3 (400MHz, CDC1₃): δ 3.73-3.81(dd, 1H, J_! = 2.8Hz, J₂ = 4Hz),

3.72 (m, 2H), 3.64-3.69 (m, 4H), 3.59 (t, 2H, J = 5.2Hz), 3.45-3.41(dd, 1H, J, = 6Hz, J₂ = 6Hz), 3.18-3.16 (m, 1H), 2.80 (t, 1H, J = 4.4Hz), 2.64-2.62 (q, 1H, Ji = 4.8Hz), 2.38 (t, 1H) 1.68 (m, 4H), 1.53-1.50 (m, 4H), 1.41 (M, 2H) [00431] ¹³CNMR (100MHz, CDCI3): δ 22.8, 25.4, 33.5, 44.1, 50.7, 59.1, 60.9,62.1 , 70.9, 71.5, 71.8, 100.0

[00432] LC-MS: 260 (M⁺)

Example 15. Synthesis of 2-[l-(2-Oxiranylmethoxy-ethoxy)-cyclopentyloxy]-

14

15

[00434] To a mixture of cyclopentanone (11) (260 g) and trimethylorthoformate (393.6 g), was added p-toluene sulfonic acid (5.88 g) in portions at 0°C. After being stirred for lhr at room temperature (there was no reaction at RT, it proceeds during heating), the reaction mass was distilled at atmospheric pressure through vigreaux column. Methyl formate and methanol were removed first, and then trimethylorthoformate. The product, compound 12 was distilled out as colorless liquid. (Boiling point 125-138°C). Yield: 315 g (80%)

[00435] 'HNMR (400MHZ, CDC1₃): δ -4.45 (s, 1H), 3.67 (s, 3H), 3.21 (s, 6H), 2.35- 2.31(m, 4H), 1.91-1.84(m, 2H), 1.76-1.74 (m, 4H), 1.67-1.65 (m, 4H). [00436] To a solution of ethylene glycol monoacetate (440 g) in THF (4.4 L), was added pyridinium p-toluene sulfonate (53.1 g), compound 12 (219.8 g) and molecular sieves (5A) (1.8 kg) sequentially and the reaction mixture was stirred at RT for 48 hours (TLC). Then sodium bicarbonate (55g) was added to the reaction mass and stirred for lOmin. Then it was filtered through celite + ₂C0₃ bed. The filtrate was evaporated at reduced pressure to obtain crude product 13, which was purified by silica gel column chromatography using 10-20% ethyl acetate / hexane as eluent. Yield: 235 g(50%)

[00437] 1HNMR (400MHz, CDC1₃): δ 4.22-4.19 (t, 4H, J = 4.8Hz), 3.67-3.65 (t, 4H, J = 4.8Hz), 2.07 (s, 6H), 1.79 (m, 4H), 1.69 (m, 4H).

[00438] ¹³CNMR (100MHz, CDC1₃): δ 20.8, 22.7, 34.4, 59.8, 63.7, 111.9, 170.8. [00439] Potassium carbonate (355.2 g) and water (120 mL) was added to a solution of compound 13 (235 g) in methanol (2.35 L). It was stirred at RT for 3 hours (TLC). Reaction mixture was filtered through celite bed, washed with methanol and evaporated the filtrate. The residue obtained was triturated with excess diethyl ether (5 x 2L) to extract the product. The combined organic layer was evaporated at reduced pressure to obtain crude product, which was washed with hexane (2 x 200ml) to furnish pure compound 14.

[00440] "HNMR (400MHz, CDC1₃): δ 3.75-3.74 (t, 4H, J - 4Hz), 3.61-3.59 (t, 4H, J = 4Hz), 2.21 (bs, 2H), 1.83-1.81 (m, 4H), 1.68-1.67(m, 4H). Yield: 160 g (50%)

[00441] ¹³CNMR (100MHz, CDC1₃): δ 22.8, 34.6, 61.9, 63.0, and 1 1 1.8.

[00442] To a solution of potassium-t-butoxide (94.37 g) in THF (2.5L) at RT, was added a solution of compound 14 (160 g) in THF (700ml) slowly and stirred for 15minutes. Then a solution of epichlorohydrin (622. 5 g) in THF (1L) was added into reaction mixture and stirred at RT for 18hours (TLC). The reaction mass was filtered through celite bed to remove inorganics. The filtrate was evaporated at reduced pressure to obtain crude product 15, which was purified by flash column chromatography on silica gel deactivated with 1% triethylamine. Product was eluted with 25-35% ethyl acetate / hexane. Yield: 62.1 g (30%)

[00443] 'H MR (400MHZ, CDC1₃): δ 3.84-3.80 (dd, 1H, Ji = 2.8Hz, J₂ = 2.8Hz), 3.74 (m, 2H), 3.70-3.69 (m, 4H), 3.62-3.59 (t, 2H, J = 4.4Hz), 3.45-3.40 (dd, 1H, Ji = 6Hz, J₂ = 6Hz), 3.18-3.16 (m, 1H), 2.81-2.79 (t, 1H, J = 4.4Hz), 2.63-2.61(q, 1H, J, = 2.8Hz, J₂ = 2.8Hz), 2.32- 2.30 (t, 1H, exchangeable) 1.82-1.80 (m, 4H), 1.69-1.67 (m, 4H).

[00444] ¹³CNMR (100MHz, CDC1₃): δ 22.7, 34.5, 43.9, 50.6, 61.0,61.9, 62.9, 70.6, 71.6, 111.7.

[00445] LC-MS: 245.0(M-1)⁺. [00446] Example 16. Synthesis of 2-(2-methyl-4-((oxiran-2-yl methoxy)methyl)-l,3- dioxolan-2-yl)ethanol (Compound 22)

[00447] To a solution of glycerol (16) (200 g) in acetone (5.6 L), was added p- toluenesulfonic acid monohydrate (10.65 g) at RT under argon atmosphere and stirred for 18Hrs (TLC). It was then neutralized with anhydrous potassium carbonate. Reaction mass was filtered through celite bed and the filtrate evaporated fully at reduced pressure. The residue obtained was re-dissolved in diethylether and washed with saturated sodium bicarbonate solution followed by brine solution. The organic layer was dried over anhydrous sodium sulfate and evaporated at reduced pressure to obtain pure compound 17. Yield: 182 g (63%) [00448] To an ice cooled suspension of NaH (81.8 g) in THF (750 mL), was added a solution of compound 17 (150 g) in THF drop wise at 0°C. It was then warmed to room temperature and then maintained for 30 minutes. It was cooled back to 0°C and then added a solution of allyl bromide (109 mL) in THF at 0°C. After addition, the reaction mass was warmed to room temperature again and stirred overnight (TLC). It was then quenched with ice water and then extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate and evaporated at reduced pressure to obtain, crude product 18, which was purified by silica gel chromatography using ethyl acetate / hexane as eluent. Yield: 148 g (75%)

[00449] To a stirred solution of 18 (148 g) in methanol (740 mL), was added water (296 mL), followed by pTSA.H₂0 (81.7 g) at RT and stirred at for lHr (TLC). Then it was neutralised by stirring for lhr with solid potassium carbonate. The reaction mass was evaporated at reduced pressure and the residue obtained was triturated with ethyl acetate. It was then filtered through celite bed and the filtrate was evaporated at reduced pressure to obtain crude product 19, which was used for the next step with out any purification. Yield: 108 g (95%)

[00450] To a stirred solution of 19 (50 g) in dichloroethane (600 mL), was added ethyl acetoacetate (38.2 g) followed by pTSA.H₂0 (2.71 g) at RT. It was then refluxed with dean-stark condenser for 3Hrs (TLC). The reaction mass was cooled to RT and neutralized with anhydrous K2CO3. It was filtered through celite bed and then washed with dichloroethane. The filtrate was evaporated at reduced pressure to obtain the crude product 20, which was purified by silica gel column chromatography using ethyl acetate in Hexane as eluent. Yield: 51 g (73%)

[00451] To an ice cooled suspension of lithium aluminiumhydride (22.8 g) in THF (735 mL), was added a solution of 20 (147 g) in THF at 0°C. Stirred at 0°C for 15 minutes and then warmed to RT. The stirring was continued for lHr to complete the reaction. It was then quenched with sat Na₂S0₄ solution at 0°C and then diluted with ethyl acetate. The reaction mass was filtered through celite bed and washed with ethyl acetate. The filtrate was separated, and the organic layer was dried over Na₂S0₄, evaporated at reduced pressure to obtain crude product 21, which was taken as such for the next step. Yield: 1 15 g (94%)

[00452] To a solution of 21 (122g) in DCM (1.22 L), was added mCPBA (174 g) portion- wise at RT and stirred overnight (TLC). It was filtered to remove any residue and the filtrate was evaporated at reduced pressure to obtain a residue. This residue was re-dissolved in DCM (500ml) and filtered to remove any undissolved material. The filtrate was evaporated at reduced pressure to obtain crude product 22 (this operation was repeated to remove chlorobenzoic acids, if any), which was purified by silica gel (deactivated by 1% triethylamine) column chromatography using ethyl acetate in hexane as eluent. Yield:68 g (52%)

[00453] Ή NMR (400MHz, CDC1₃): δ 1.43 (d, 3H), 1.95 (m, 2H), 2.61 (m, 1H), 2.80 (t, 1H), 3.17 (m, 1H), 3.44 (m, 1H), 3.65 (m, 2H), 3.86 (m, 4H), 4.13 (m, 1H), 4.31 (m, 1H). [00454] ¹³C NMR (100MHz, CDC1₃): δ 23.97, 23.99, 24.76, 40.22, 40.60, 43.88, 50.52, 50.59, 50.62, 58.38, 58.80, 66.13, 66.43, 66.46, 71.21, 71.39, 71.85, 72.06, 72.13, 74.1 1, 74.20, 75.17, 75.24, 111.15

[00455] LC-MS: 241 (M+23) ⁺.

[00456] Example 17. Synthesis of (8-(Oxiran-2-ylmethoxy)-l,4-dioxaspiro [4.5] decan 2-yl) methanol (Compound 27)

[00457] To a solution of 1 ,4-cyclohexanedione monoethylene ketal (23) (125 g) in water (625 mL) at 0°C, was added sodium borohydride (15.13 g) as 10 portions. Then the reaction mixture was warmed to RT and stirred for 1 hour (TLC). It was then saturated with NaCl and extracted with EtOAc several times to recover the product completely from aqueous layer. The organic layer was dried over anhydrous sodium sulfate and evaporated at reduced pressure to obtain pure product 24. Yield: 125 g (97%)

[00458] To a suspension of sodium hydride (60% in paraffin oil, 50.57 g) in THF (1 L) at 0°C, was added compound 24 (100 g) in THF and stirred for 15 minutes. Then a solution of allyl bromide (91.76 g) in THF was added to the reaction mixture at 0°C. The reaction mass was then warmed to RT and stirred for 4 hours (TLC). It was cooled to 0°C, quenched with ice water and extracted with diethyl ether to obtain the allylated product. This ethereal solution was cooled to 0°C, added 2N HCl and stirred for 1 hour at RT. The layers were separated and the organic layer was dried over anhydrous sodium sulfate and evaporated to afford the cyclohexanone derivative 25 as pure product. Yield: 80 g (80%)

[00459] To a stirred solution of glycerol (140 g) in THF (1.4 L), was added p-Toluene sulfonic acid (14.5 g), compound 25 (93.5 g) followed by 4A molecular sieves (560 g) sequentially. It was stirred at room temperature for 15 hours (TLC). Then sodium bicarbonate (14.5g) was added to the reaction mass and stirred for 10 minutes. It was then filtered through celite pad having anhydrous K₂C0₃ on the top. The filtrate was then evaporated at reduced pressure to obtain crude product, which was dissolved in water and extracted with ethyl acetate. The organic layer was again washed with water to remove glycerol completely, dried over anhydrous sodium sulfate and evaporated at reduced pressure to afford pure product 26. Yield: 120g (85%)

[00460] To a solution of 26 (120 g) in DCM (1.2 L), was added m-chloroperbenzoic acid ( 181.5 g) and stirred at RT for 15hours (TLC). Then the white suspension of the reaction mass was filtered to remove m-chlorobenzoic acid. The filtrate was evaporated at reduced pressure, and the resultant residue was again suspended in DCM and filtered to remove the acid. The filtrate was evaporated at reduced pressure as such to obtain crude product 27, which was purified by silica gel (deactivated with 1% triethylamine) column chromatography using 50% EtOAc / hexane as eluent. Yield: 63 g (50%)

[00461] 'H MR (400MHz, CDC1₃): δ 4.25-4.21 (m, 1H), 4.10-4.02 (m, 1H), 3.81-3.77 (m, 3H), 3.72-3.71 (dd, 1H, J = 1.2Hz), 3.43 (m, 2H), 3.15-3.14 (d, 1H, J = 2.8Hz), 2.81-2.77 (t, 1H, J = 4.8Hz), 2.63-2.62 (t, 1H, J = 2.0Hz), 2.05 (m, 1H), 1.86-1.81 (m, 6H), 1.69-1.62 (m, 2H).

[00462] ¹³CNMR (100MHz, CDC1₃): δ -28.1, 30.7, 32.3, 33.8, 44.2, 50.9, 62.8, 65.4, 68.5, 74.5, 75.7, 108.9.

[00463] Example 18. Synthesis of PKHA copolymer by copolymerization of compound 10 and glycidol

[00464] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.090 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 95°C and a mixture of the ketal monomer 10 (3.4 g, 0.0131 moles) and glycidol (0.28 mL, 0.0042 moles) was added over a period of 12 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 12 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum. [00465] Mn (from GPC in chloroform) (Figure 29): 22 000

[00466] Mw/Mn : 1.34

[00467] NMR analysis showed that the copolymer contains 60 mol% of ketal groups

(Figure 25).

[00468] The polymer was insoluble in water, but soluble in organic solvents such as methanol, chloroform, dichloromethane, THF, and DMF etc. In order to make the polymer water-soluble, 20-25 mol% of the hydroxyl groups were modified using MPEG-epoxide-400.

[00469] Example 19. Synthesis of PKHA copolymer by copolymerization of compound 10 and glycidol

[00470] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added 1,1,1 -tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.090 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 95°C and a mixture of the ketal monomer 10 (8 g, 0.0308 moles) and glycidol (0.68 mL, 0.0105moles) was added over a period of 12 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 12 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum.

[00471] Mn (from GPC in chloroform): 56 700

[00472] Mw/Mn : 1.6

[00473] NMR analysis showed that the copolymer contains 65 mol% of ketal groups.

[00474] The polymer was insoluble in water, but soluble in organic solvents such as methanol, chloroform, dichloromethane, THF, and DMF etc. In order to make the polymer water-soluble, 20-25 mol% of the hydroxyl groups were modified using MPEG-epoxide-400. [00475] Example 20. Synthesis of PKHA copolymer by copolymerization of compound 15 and glycidol

[00476] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.090 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 95°C and a mixture of the ketal monomer 15 (3.6 g, 0.0146 moles) and glycidol (0.3 mL, 0.0049 moles) was added over a period of 12 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 12 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum.

[00477] Mn (from GPC in chloroform): 7 600 [00478] Mw/Mn : 1.6

[00479] NMR analysis showed that the copolymer contains 58 mol% of ketal groups.

[00480] The polymer was insoluble in water, but soluble in organic solvents such as methanol, chloroform, dichloromethane, THF, and DMF etc. In order to make the polymer water-soluble, 20-25 mol% of the hydroxyl groups were modified using MPEG-epoxide-400.

[00481] Example 21. Synthesis of PKHA copolymer by copolymerization of compound 15 and glycidol

[00482] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l ,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.090 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 95 °C and a mixture of the ketal monomer 15 (8 g, 0.0325 moles) and glycidol (0.72 mL, 0.011 moles) was added over a period of 12 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 12 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum.

[00483] Mn (from GPC in chloroform) (Figure 30): 14000 [00484] Mw/Mn : 1.65

[00485] NMR analysis showed that the copolymer contains 62 mol% of ketal groups

(Figure 26).

[00486] The polymer was insoluble in water, but soluble in organic solvents such as methanol, chloroform, dichloromethane, THF, and DMF etc. In order to make the polymer water-soluble, 20-25 mol% of the hydroxyl groups were modified using MPEG-epoxide-400.

[00487] Example 22. Synthesis of PKHA copolymer by copolymerization of compound 22 and glycidol

[00488] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added 1,1,1 -tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.090 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 95°C and a mixture of the ketal monomer 22 (7 g, 0.0321 moles) and glycidol (0.7 mL, 0.0108 moles) was added over a period of 12 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 12 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum.

[00489] Mn (from GPC in chloroform) (Figure 31): 7 000 [00490] Mw/Mn : 1.6

[00491] NMR analysis showed that the copolymer contains 57 mol% of ketal groups

(Figure 27).

[00492] The polymer was insoluble in water, but soluble in organic solvents such as methanol, chloroform, dichloromethane, THF, and DMF etc. In order to make the polymer water-soluble, 20-25 mol% of the hydroxyl groups were modified using MPEG-epoxide-400.

[00493] Example 23. Synthesis of PKHA copolymer by copolymerization of compound 27 and glycidol

[00494] In a three-necked round-bottomed flask cooled under vacuum and filled with argon was added l,l,l-tris(hydroxymethyl)propane (TMP, 0.048 g). To this potassium methylate (25% solution in methanol, 0.090 mL) was added and stirred under argon for 30 minutes.

Methanol was removed under vacuum for 3 h. The flask was heated to 95°C and a mixture of the ketal monomer 27 (7 g, 0.0287 moles) and glycidol (0.62 mL, 0.0096 moles) was added over a period of 12 h using a syringe pump. After complete addition of monomers, the reaction mixture was stirred for an additional 12 h. The polymer was dissolved in methanol, precipitated from diethyl ether and dried under vacuum.

[00495] Mn (from GPC in chloroform): 8 600

[00496] Mw/Mn : 1.58

[00497] NMR analysis showed that the copolymer contains 45 mol% of ketal groups

(Figure 28).

[00498] The polymer was insoluble in water, but soluble in organic solvents such as methanol, chloroform, dichloromethane, THF, and DMF etc. In order to make the polymer water-soluble, 20-25 mol% of the hydroxyl groups were modified using MPEG-epoxide-400.

[00499] Example 24. Degradation kinetics of poly(ketal hydroxyalkanes) at different pHs

[00500] The kinetics of degradation of the polymers was studied by dissolving the polymer in different buffers in D₂0 and monitoring by NMR the disappearance of peak corresponding to the ketal group and consequent appearance of new peaks due to formation of the respective ketone. Aqueous solutions of poly (ketal hydroxyalkanes) polymer were prepared in different buffers in D₂0 and the degradation was analyzed (Figures 32-41).

[00501] PKHA polymers synthesized in Example 14, 19 and 22 bearing dimethyl, cyclohexyl and cyclopentyl ketal groups respectively exhibited good pH dependent degradation profiles in the pH range 5.5-7.4 at 25°C. However, PKHA polymers in Example 23 and 24 did not show any degradation at 25°C in this pH range even after 90 days, a slow degradation was observed at pH 4.1. So the degradation of these two polymers was also studied at 37°C.

[00502] Example 25. Amine-functionalization and fluorophore-labeling of PKHA [00503] PKHA copolymer synthesized in Example 13 (0.30 g) and NaH (15 mg) were stirred in 5 mL of 1 -methyl 2-pyrrolidinone (NMP) at 25°C for 5 h. MPEG-epoxide-400 (0.15 g) dissolved in NMP was added and the reaction mixture was stirred at 70°C for 15 h. Azido- (PEG)g-epoxide (0.040 g) was then added and the reaction mixture stirred at 70°C for another 15 h. The reaction mixture was cooled to RT, the polymer was precipitated twice from diethyl ether and dried under vacuum to obtain the azide-functionalized polymer. Reduction of the azide group to amine was carried out using Ph₃P/H₂0 in THF at 25°C for 24 h. The polymer was then precipitated from diethyl ether and dried under vacuum.

[00504] The amine-functionalized polymer was reacted with N-hydroxy succinimide ester of Alexa-488 carboxylic acid to afford the fluorophore-labeled polymer.

[00505] Example 26. Amine-functionalization and fluorophore-labeling of PKHA

[00506] PKHA copolymer synthesized in Example 18 (0.30 g) and NaH (15 mg) were stirred in 5 mL of 1 -methyl 2-pyrrolidinone (NMP) at 25°C for 5 h. MPEG-epoxide-400 (0.13 g) dissolved in NMP was added and the reaction mixture was stirred at 70°C for 15 h. Azido- (PEG)₈-epoxide (0.040 g) was then added and the reaction mixture stirred at 70°C for another 15 h. The reaction mixture was cooled to RT, the polymer was precipitated twice from diethyl ether and dried under vacuum to obtain the azide-functionalized polymer. Reduction of the azide group to amine was carried out using Ph₃P/H₂0 in THF at 25°C for 24 h. The polymer was then precipitated from diethyl ether and dried under vacuum. [00507] The amine-functionalized polymer was reacted with N-hydroxy succinimide ester of Alexa-488 carboxylic acid to afford the fluorophore-labeled polymer.

[00508] Example 27. Amine-functionalization and fluorophore-labeling of PKHA

[00509] PKHA copolymer synthesized in Example 28 (0.30 g) and NaH (15 mg) were stirred in 5 mL of 1 -methyl 2-pyrrolidinone (NMP) at 25°C for 5 h. MPEG-epoxide-400 (0.15 g) dissolved in NMP was added and the reaction mixture was stirred at 70°C for 15 h. Azido-

(PEG)g-epoxide (0.10 g) was then added and the reaction mixture stirred at 70°C for another 15 h. The reaction mixture was cooled to RT, the polymer was precipitated twice from diethyl ether and dried under vacuum to obtain the azide-functionalized polymer. Reduction of the azide W group to amine was carried out using PI13P/H2O in THF at 25°C for 24 h. The polymer was then precipitated from diethyl ether and dried under vacuum.

[00510] The amine-functionalized polymer was reacted with N-hydroxy succinimide ester of Alexa-488 carboxylic acid to afford the fluorophore-labeled polymer. [00511 ] Example 28: Conjugation of siRNA to polyketal polymers

[00512] A general scheme for conjugation of siRNA to polyketal polymers is as shown in Figure 42.

[00513] Conjugation of siRNA to polymer: 0.32 mg of siRNA dissolved in 50 of phosphate buffer at pH 8.5 and 0.85 mg of AR-106 (ketal polymer) in 150 μί of phosphate buffer at pH 8.5 was added to the mixture. The reaction mixture was stirred for 4 hrs at room temperature. The mixture was injected to GPC and analyzed using the same conditions used for the ketal polymers.

[00514] Polyketals degrade very fast under acidic conditions. The polyketals were modified with azide groups (2 per chain). The alkyne functionalized siRNAs were reacted with azide by copper free click chemistry. The conjugates were then characterized by GPC coupled with UV and light scattering detectors. The conjugate formation is confirmed by increase in molecular weight and disappearance of free siRNA peak. The formation of conjugate is clearer from the UV traces of polymer and polymer-siRNA conjugate.

[00515] No significant degradation was observed within a week if the polymer alone was stored in solution at pH 8.5. However the siRNA conjugate was found to degrade within 3-4 days. Accordingly, each conjugate was prepared and tested within a day where possible, for example for the dimethyl ketal polymer. This compound may however be stable at low temperature. Results are shown in Figures 43-45.

[00516] Conjugation and characterization of dimethyl Ketal polymers [00517] Example 28a:

Polymer AR-105 36 1.8

Conjugate RKK-299A 31425 50 2.4 2.6

Conjugate RKK-299C 31426 52 2.5 2.1

Polymer AR-106 80 1.5

Conjugate RKK-299B 31425 90 1.6 2.7

Conjugate RKK-299D 31426 72 2.1 2.9

[00518] Free uptake and silencing in mouse primary hepatocytes

[00519] Example 28b.

Sample No siRNA Molecular Polydispersity Concentration weight kD (μΜ)

Polymer AR-105 36 1.8

Conjugate RKK-304-A 31426 40 2.2 1 10

Polymer AR-106 80 1.5

Conjugate RKK-304-B 31426 82 1.5 1 10

Control siRNA RKK-304-C 31426 160 (alkyne modified

with a small molecule

azide [00520] Free uptake and silencing experiment in mouse primary hepatocytes

SEQ ID NO: 35 (AD-31426, sense); SEQ ID NO: 36 (AD-31426, anti-sense); SEQ ID NO: 37(AD-27813, sense); SEQ ID NO: 38(AD-27813, anti-sense)

[00521 ] All citations are herein incorporated by reference, as if each individual publication was specifically and individually indicated to be incorporated by reference herein and as though it were fully set forth herein. Citation of references, patents, patent applications or patent publications herein is not to be construed nor considered as an admission that such references are prior art to the present invention.

Claims

WHAT IS CLAIMED IS:

1. A polymer according to Formula (I):

(I)

wherein

and combinations thereof; wherein the polymer comprises at least one of

wherein Ri and R₂ are each independently hydrogen, alkyl, substituted alkyl, or Ri and R₂ can be taken together with the atom they are attached to form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl;

Y and Z are each independently H, OH or

, where v is 1-10; Ri₀ and R₂o are each independently H, or Formula II;

R is selected from hydrogen, N₃, -NH₂, -NH, COOH, NHS ester, alkene, alkyne, aldehyde, maleimide, SH, polyethylene glycol (PEG) of varying length and/or MW and -J-W; where J is absent or a linker and W is a therapeutic agent, a targeting ligand, an endosomolytic ligand, a PK (pharmacokinetic) modulator, Z, Y, or a combination thereof.

A polymer according to Formula (II):

(ID

wherein

X is absent or is selected from C, O, S, alkyl, alkenyl, alkynyl, aryl, substituted alkyl, substituted alkenyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl, heterocycles with at least one heteroatom in the ring, N(R), S-S, C(0)0, C(0)N(R), N(R)C(0)0, N(R)C(0)N(R), OC(O), OC(0)N(R), N(R)C(0), N(R)C(0)N(R), N(R)C(S)N(R), OC(S)N(R), N(R)C(0) or N(R)C(S)0;

R_\ and R₂ are each independently hydrogen, alkyl, substituted alkyl, or R_\ and R₂ can be taken together with the atom they attached to form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl; r, s, and t are each independently 0-10,000; q is 5-30,000; pi to p7 are each independently 0-10;

Y and Z are each independently H, OH or

, where v is 1-10; R 10 and R₂o are each independently H, or formula II;

R is selected from hydrogen, N₃, -NH₂, -NH, COOH, NHS ester, alkene, alkyne, aldehyde, maleimide, SH, PEG of varying length and/or MW and -J-W; where J is absent or a linker and W is a therapeutic agent, a targeting ligand, an endosomolytic ligand, a PK (pharmacokinetic) modulator, Z, Y, or a combination thereof.

3. A polymer according to formula (III):

(III)

wherein X is absent or is selected from C, O, S, alkyl, alkenyl, alkynyl, aryl, substituted alkyl, substituted alkenyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl, heterocycles with at least one heteroatom in the ring, N(R), S-S, C(0)0, C(0)N(R), N(R)C(0)0, N(R)C(0)N(R), OC(O), OC(0)N(R), N(R)C(0), N(R)C(0)N(R), N(R)C(S)N(R), OC(S)N(R), N(R)C(0) or N(R)C(S)0;.

Ri and R₂ are each independently alkyl, substituted alkyl, or Ri and R₂ can be taken together with the atom they attached to form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl; r, s, and t are each independently 0-10,000;

q is 5-30,000;

pi to p7 are each independently 0-10;

where v is 1-10; Rio and R₂₀ are each independently H, or formula II;

R is selected from hydrogen, N₃, -NH₂, -NH, COOH, NHS ester, alkene, alkyne, aldehyde, maleimide, SH, PEG of varying length and/or MW and -J-W; where J is absent or a linker and W is a therapeutic agent, a targeting ligand, an endosomolytic ligand, a PK (pharmacokinetic) modulator, Z, Y or a combination thereof.

4. A polymer according to formula (IV): (IV)

wherein

X is absent or is selected from C, O, S, alkyl, alkenyl, alkynyl, aryl, substituted alkyl, substituted alkenyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl, heterocycles with at least one heteroatom in the ring, N(R), S-S, C(0)0, C(0)N(R), N(R)C(0)0, N(R)C(0)N(R), OC(O), OC(0)N(R), N(R)C(0), N(R)C(0)N(R), N(R)C(S)N(R), OC(S)N(R),

N(R)C(0) or N(R)C(S)0;

Ri and R₂ are each independently alkyl, substituted alkyl, or Rj and R₂ can be taken together with the atom they attached to form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl; q is 5-30,000; pi to p4 are each independently 0-10; nl to n2 each independently 0-10,000;

Y and Z are each independently H, OH or

, where v is 1-10;

Rio and R₂₀ are each independently H, or Formula II;

5. A polymer according to Formula (V):

(V)

wherein

N(R)C(0) or N(R)C(S)0;

Ri and R₂ are each independently hydrogen, alkyl, substituted alkyl, or Ri and R₂ can be taken together with the atom they attached to form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl; r, s, and t are each independently 1-10,000; q is 5-30,000; v is 1-10; pi to p21 are each independently 0-10; ml, ml or m3 each independently 1-20;

R is selected from hydrogen, N₃, -NH , -NH, COOH, NHS ester, alkene, alkyne, aldehyde, maleimide, SH, PEG of varying length and/or MW and -J-W; where J is absent or a linker and W is a therapeutic agent, a targeting ligand, an endosomolytic ligand, a PK (pharmacokinetic) modulator, or a combination thereof;

Yl, Y2 and Y3 are each independently H, OH or

where v is 1-

10; and

Rio and R₂₀ are each independently H, or formula II;

6. The polymer of any one of claims 1 to 5, wherein the polymer is a homopolymer, random copolymer, tercopolymer, block copolymer, linear, branched, dendritic, amphiphilic, matrix, block or crossed-linked polymer.

7. The polymer of any one of claims 1 to 5, wherein the endosomolytic ligand is selected from the group consisting of imidazoles, poly or oligoimidazoles, linear or brached polyethyleneimines (PEIs), linear and branched polyamines, cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals, polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, and natural and synthetic fusogenic lipids.

8. The polymer of any one of claims 1 to 5 wherein the endosomolytic ligand is a polyanionic peptide or a polyanionic peptidomimetic.

9. The polymer of any one of claims 1 to 5 wherein the endosomolytic ligand is selected from the group consisting of GALA, EALA, INF-7, Inf HA-2, diINF-7, diINF3, GLF, GALA-INF3, INF-5, JTS-1, ppTGl, ppTG20, KALA, HA, melittin, and histidine-rich peptide CHK^HC.

I l l 10. The polymer of any one of claims 1 to 5 wherein the targeting ligand is selected from the group consisting of an antibody, a ligand-binding portion of a receptor, a ligand for a receptor, an aptamer, D-galactose, N-acetyl-D-galactose (GalNAc), multivalent N-acetyl-D- galactose, D-mannose, cholesterol, a fatty acid, a lipoprotein, folate, thyrotropin,

melanotropin, surfactant protein A, mucin, carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate,

polyaspartate, a lipophilic moiety that enhance plasma protein binding, a steroid, bile acid, vitamin B₁₂, biotin, an RGD peptide, an RGD peptide mimetic, ibuprofen, naproxen, aspirin, folate, and analogs and derivatives thereof.

11. The polymer of any one of claims 1 to 5, wherein the targeting ligand is selected from the group consisting of D-galactose, N-acetyl-D-galactose (GalNAc), multivalent N-acetyl-D- galactose, cholesterol, folate, and analogs and derivates thereof.

12. A composition comprising the polymer of any one of claims 1 to 11 , and a nucleic acid.

13. The composition of claim 12 wherein the nucleic acid is selected from the group consisting of an iRNA agent, an antisense oligonucleotide, an antagomir, an activating RNA, a decoy oligonucleotide, an aptamer, and a ribozyme.

14. The composition of claim 12, wherein the nucleic acid is non-covalently linked to the polymer.

15. The composition of claim 12 wherein the nucleic acid is covalently linked to the polymer.

16. The composition of claim 12, wherein the nucleic acid decreases expression of a target gene through an RNA interference mechanism.

17. The composition of claim 12 wherein the nucleic acid is a single-stranded

oligonucleotide. 18. The composition of claim 12 wherein the nucleic acid is a double-stranded oligonucleotide.

19. The polymer of any one of claims 1 to 5 wherein the linker is a redox cleavable linker.

20. The polymer of any one of claims 1 to 5 wherein the linker comprises a pH sensitive component.

21. A method of delivering a nucleic acid to a cell, the method comprising (a) contacting a cell with the composition of claim 12; and (b) allowing the cell to internalize the polymer.

22. The method of claim 21 where the nucleic acid is retained within the cell.

23. A method of inhibiting expression of one or more gene products, the method comprising contacting one or more cells with a polymer according to one or more of Formula I, II, III, IV or V, in combination with an amount of nucleic acid effective to decrease expression of one or more gene products.