WO2017112852A1

WO2017112852A1 - Modified polyethyleneimines and uses thereof

Info

Publication number: WO2017112852A1
Application number: PCT/US2016/068260
Authority: WO
Inventors: Sonke Svenson; Richard W. Heidebrecht; Victor Kamhi; Jungyeon Hwang; Snehlata Tripathi
Original assignee: Preceres Inc.
Priority date: 2015-12-23
Filing date: 2016-12-22
Publication date: 2017-06-29

Abstract

The present disclosure is directed to modified polyethyleneimines, formulations thereof further comprising at least one active agent, as well as methods of delivering the at least one active agent to a target organism.

Description

MODIFIED POLYETHYLENEIMINES AND USES THEREOF

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial

No. 62/387,296, filed December 23, 2015, which is hereby incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present disclosure relates generally to novel modified polyethyleneimines

(i.e., modified PEIs). The present disclosure also relates generally to methods of preparing and screening such modified PEIs, their formulation with one or more active agents, and the delivery of such formulations to target organisms.

BACKGROUND OF THE INVENTION

[0003] A wide range of molecules have been employed for delivering polynucleotides and other active agents to cells. In particular, polymers such as polyethylenimine (i.e., PEI) or poly(beta-aminoesters) have been used to effectively complex DNA for delivery into cells. Polymers in these classes of delivery agent typically contain amine functionalities that serve to electrostatically bind to DNA to form nanoparticles that are then taken up by the cell via endocytosis. Once in the cell, these amine groups serve to buffer the endosome and cause an influx of ions due to the proton-sponge mechanism. The resulting burst of the endocytic vesicle leads to the release of the payload of the particle, which is then free to travel to the nucleus where the DNA is expressed.

[0004] While such polymer based systems have been used extensively for DNA delivery, the delivery of other molecules, such as RNA, presents distinct challenges. In many cases, polymeric materials do not work as effectively for RNA delivery. This is likely due to differences in the chemical structure of the RNA being delivered compared to DNA. RNA are generally short, linear fragments containing additional hydroxyl moieties on each ribose ring. These differences necessitate an alternative approach that is suited for complexation with short RNA strands. In particular, an improved delivery system is required for the use of RNA for agricultural and pharmaceutical applications. The delivery system needs to protect RNA from nuclease degradation, allow for the proper concentration and distribution profile in the target tissues, facilitate efficient uptake of RNA into target cells, and release RNA into cytoplasm to knockout expression of the target gene.

[0005] Particular drawbacks to using a PEI as the payload delivery agent include the cytotoxicity of certain of these polymers, which has been reported to increase as the molecular weight of the PEI increases, as well as their relatively poor aqueous solubility. Another drawback is the high polydispersity index of commercially available PEIs, which contributes to batch-to-batch variability in micro- and nanoparticle formulations formed from such PEIs and active agents.

[0006] Thus, there exists a continuing need for PEI-based delivery agents that possess an improved ability to deliver DNA and RNA, as well as other active agents, to cells over existing PEI delivery agents. In particular, there is a continuing need for PEI-based delivery agents that exhibit decreased cytotoxicity, improved aqueous solubility, and improved batch consistency when formulated with active agents into micro- and nanoparticles.

EMBODIMENTS OF THE INVENTION

[0007] One embodiment of the present invention is a modified polyethyleneimine comprising a structural unit of formula (I) and/or formula (II):

wherein: is hydrogen, a group of formula (III):

wherein R₅ is hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted hetero aliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group; and

R₆ is an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted hetero aliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group; a group of formula (IV):

wherein

are, independently, hydrogen or an optionally substituted aliphatic group; R9 is an optionally substituted aliphatic group; and

X is O or NR11, wherein Rn is selected from the group consisting of H and Ci to C10 alkyl; or a group of formula (V):

wherein

Rio is selected from the group consisting of Ci to C₄ alkyl groups;

are, independently, hydrogen, a group of formula (III):

wherein R₅ and R₆ are as defined above; or a group of formula (IV):

wherein R₇, Rg, R9, and X are as defined above; wherein the nitrogen atom bonded to Ri in formula (I) and the nitrogen atom bonded to R₄ in formula (II) can each, independently, instead be bonded to an ethylene moiety in the modified polyethyleneimine; and with the proviso that at least one of Ri, R₂, R₃, and R₄ is a group of formula (III) or a

group of formula (IV).

[0008] Another embodiment of the present invention is the above modified polyethyleneimine, wherein at least one of Ri, R₂, R₃, and R₄ is a group of formula (III).

[0009] Another embodiment of the present invention is the above modified polyethyleneimine, wherein at least Ri and R₂ are each, independently, a group of formula (III).

[0010] Another embodiment of the present invention is the above modified polyethyleneimine, wherein at least Ri, R₂, and R₃ are each, independently, a group of formula (III).

[0011] Another embodiment of the present invention is the above modified polyethyleneimine, wherein Ri, R₂, R₃, and R₄ are each, independently, a group of formula (III). [0012] Another embodiment of the present invention is the above modified polyethyleneimine, wherein R₆ is a Ci to C₂0 alkyl group.

[0013] Another embodiment of the present invention is the above modified polyethyleneimine, wherein R₆ is a C₄ to C₁₆ alkyl group.

[0014] Another embodiment of the present invention is the above modified polyethyleneimine, wherein R₆ is a C₆ to C₁₄ alkyl group.

[0015] Another embodiment of the present invention is the above modified polyethyleneimine, wherein R5 is H and R₆ is selected from the group consisting of groups of formulae (XI) through (XXVII):

[0016] Another embodiment of the present invention is the above modified polyethyleneimine, wherein at least one of Ri, R₂, R₃, and R4 is a group of formula (IV).

[0017] Another embodiment of the present invention is the above modified polyethyleneimine, wherein at least Ri and R₂ are each, independently, a group of formula (IV).

[0018] Another embodiment of the present invention is the above modified polyethyleneimine, wherein at least Ri, R₂, and R₃ are each, independently, a group of formula (IV). [0019] Another embodiment of the present invention is the above modified polyethyleneimine, wherein Ri, R₂, R₃, and R4 are each, independently, a group of formula (IV).

[0020] Another embodiment of the present invention is the above modified polyethyleneimine, wherein R9 is a Ci to C₂o alkyl group.

[0021] Another embodiment of the present invention is the above modified polyethyleneimine, wherein R9 is a C₈ to Ci₆ alkyl group.

[0022] Another embodiment of the present invention is the above modified polyethyleneimine, wherein R₇ and R₈ are each hydrogen,_, X is O, and R9 is a group selected from the group consisting of formulae (XXVIII), (XXIX), and (XXX):

(XXX).

[0023] Another embodiment of the present invention is the above modified polyethyleneimine, wherein R₇ and R₈ are each hydrogen, X is NH, and R9 is a group selected from the group consisting of formulae (XXIX) and (XXX):

(XXX).

[0024] Another embodiment of the present invention is the above modified polyethyleneimine, wherein the modified polyethyleneimine is branched. [0025] Another embodiment of the present invention is the above modified polyethyleneimine, wherein the modified polyethyleneimine is hyperbranched.

[0026] Another embodiment of the present invention is the above modified polyethyleneimine, wherein the modified polyethyleneimine is dendritic.

[0027] Another embodiment of the present invention is the above modified polyethyleneimine, wherein the modified polyethyleneimine is derived from a branched polyethyleneimine having a number average molecular weight (M_n) in the range of from 600 to 10,000 Daltons.

[0028] Another embodiment of the present invention is the above modified polyethyleneimine, wherein the branched polyethyleneimine has a number average molecular weight (M_n) of greater than 2,000 Daltons.

[0029] Another embodiment of the present invention is the above modified polyethyleneimine, wherein the branched polyethyleneimine has a number average molecular weight (M_n) selected from the group consisting of 600, 1,200, 1,800, and 10,000 Daltons.

[0030] Another embodiment of the present invention is the above modified polyethyleneimine, wherein the modified polyethyleneimine is derived from a branched polyethyleneimine comprising primary, secondary, and tertiary amino groups present in a ratio of 1:2: 1.

[0031] Another embodiment of the present invention is the above modified polyethyleneimine, wherein the modified polyethyleneimine is linear.

[0032] Another embodiment of the present invention is the above modified polyethyleneimine, wherein the modified polyethyleneimine is derived from a linear polyethyleneimine having an average molecular weight (M_n) of greater than 2,000 Daltons.

[0033] Another embodiment of the present invention is the above modified polyethyleneimine, wherein the linear polyethyleneimine has a number average molecular weight (M_n) in the range of from 2,500 to 100,000 Daltons. [0034] Another embodiment of the present invention is the above modified polyethyleneimine, wherein the polyethyleneimine has a number average molecular weight (M_n) of 2,500 Daltons.

[0035] Another embodiment of the present invention is the above modified polyethyleneimine, wherein the modified polyethyleneimine is derived from the partial hydrolysis of a poly(2-alkyl-2-oxazoline).

[0036] Another embodiment of the present invention is the above modified polyethyleneimine, wherein the poly(2-alkyl-2-oxazoline) is poly(2-ethyl-2-oxazoline).

[0037] Another embodiment of the present invention is the above modified polyethyleneimine, wherein Ri is a group of formulae (III), (IV), or (V), R₂ and/or R₃ is a group of formulae (III) or (IV), and R₄ is a group of formulae (III) or (IV).

[0038] Another embodiment of the present invention is the above modified polyethyleneimine, wherein Ri and R₄ are each a group of formulae (III) or (IV), and R₂ and/or R₃ is a group of formulae (III) or (IV).

[0039] Another embodiment of the present invention is the above modified polyethyleneimine, wherein Ri and R₄ are each a group of formulae (III) and R₂ and/or R₃ is a group of formulae (III).

[0040] Another embodiment of the present invention is the above modified polyethyleneimine, wherein Ri and R₄ are each a group of formulae (IV) and R₂ and/or R₃ is a group of formulae (IV).

[0041] Another embodiment of the present invention is the above modified polyethyleneimine, wherein each Ri and R₄ is independently selected from at least two different groups of formulae (III) and/or (IV), and each R₂ and/or R₃ is independently selected from at least two different groups of formulae (III) and/or (IV).

[0042] Another embodiment of the present invention is the above modified polyethyleneimine, wherein each Ri and R₄ is independently selected from at least two different groups of formulae (III), and each R₂ and/or R₃ is independently selected from at least two different groups of formulae (III). [0043] Another embodiment of the present invention is the above modified polyethyleneimine, wherein each Ri and R4 is independently selected from at least two different groups of formulae (IV), and each R₂ and/or R₃ is independently selected from at least two different groups of formulae (IV).

[0044] Yet another embodiment of the present invention is a modified

polyethyleneimine selected from the group consisting of compounds of formulae (1) through (299):

ιζ

,

the number average molecular weight of the unmodified

polyethyleneimine prior to modification;

L/B denotes whether the modified polyethyleneimine is linear or branched;

R is the ratio of modified to unmodified primary and secondary amino groups in the modified polyethyleneimine; and

C is the structure of moiety or moieties with which the structure of the polyethyleneimine has been modified.

[0045] Yet another embodiment of the present invention is a microparticle or nanoparticle comprising the above modified polyethyleneimine and an active agent to be delivered.

[0046] Another embodiment of the present invention is the above microparticle or nanoparticle, wherein the active agent to be delivered is selected from the group consisting of polynucleotides, oligonucleotides, proteins, peptides, and small molecules. [0047] Another embodiment of the present invention is the above microparticle or nanoparticle, wherein the active agent to be delivered is an oligonucleotide or a

polynucleotide.

[0048] Another embodiment of the present invention is the above microparticle or nanoparticle, wherein the oligonucleotide or polynucleotide is modified.

[0049] Another embodiment of the present invention is the above microparticle or nanoparticle, wherein the oligonucleotide or polynucleotide is unmodified.

[0050] Another embodiment of the present invention is the above microparticle or nanoparticle, wherein the active agent to be delivered is an RNA.

[0051] Another embodiment of the present invention is the above microparticle or nanoparticle, wherein the RNA is a single- stranded RNA.

[0052] Another embodiment of the present invention is the above microparticle or nanoparticle, wherein the RNA is a double- stranded RNA.

[0053] Another embodiment of the present invention is the above microparticle or nanoparticle, wherein the RNA is a small interfering RNA (siRNA).

[0054] Another embodiment of the present invention is the above microparticle or nanoparticle, wherein the RNA is a messenger RNA (mRNA).

[0055] Yet another embodiment of the present invention is a formulation comprising the above modified polyethyleneimine and a first active agent to be delivered.

[0056] Another embodiment of the present invention is the above formulation, wherein the first active agent to be delivered and the at least one modified polyethyleneimine are non-covalently associated to one another to form a non-covalent complex.

[0057] Another embodiment of the present invention is the above formulation, further comprising at least one additional active agent to be delivered.

[0058] Another embodiment of the present invention is the above formulation, wherein the at least one additional active agent to be delivered is contained within or on the surface of the non-covalent complex. [0059] Another embodiment of the present invention is the above formulation, wherein the at least one active additional agent to be delivered is not contained within or on the surface of the non-covalent complex.

[0060] Another embodiment of the present invention is the above formulation, further comprising one or more excipients.

[0061] Another embodiment of the present invention is the above formulation, wherein the one or more excipients is selected from the group consisting of fillers, extenders, binders, humectants, disintegrants, plasticizers, stabilizers, solution retarding agents, wetting agents, suspending agents, thickening agents, absorbents, lubricants, surfactants, buffering agents, diluents, solvents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, opacifying agents, separating agents, and coating permeability adjusters.

[0062] Another embodiment of the present invention is the above formulation, wherein the one or more excipients is selected from the group consisting of sterols, carbohydrates, proteins, lipids, water-soluble polymers, and any combination thereof.

[0063] Another embodiment of the present invention is the above formulation, wherein the one or more excipients comprises cholesterol.

[0064] Another embodiment of the present invention is the above formulation, wherein the one or more excipients comprises a polyethylene glycol, a polypropylene oxide, a polyvinylpyrrolidone, a polyvinyl alcohol, polylactic acid, poly(lactic-co-glycolic acid), or any combination thereof.

[0065] Another embodiment of the present invention is the above formulation, wherein the first active agent to be delivered is an oligonucleotide or a polynucleotide.

[0066] Another embodiment of the present invention is the above formulation, further comprising an agriculturally acceptable carrier.

[0067] Another embodiment of the present invention is the above formulation, wherein the oligonucleotide or polynucleotide modulates the expression of a gene in a plant. [0068] Another embodiment of the present invention is the above formulation, wherein the oligonucleotide or polynucleotide modulates the expression of a gene in an insect.

[0069] Another embodiment of the present invention is the above formulation, wherein the oligonucleotide or polynucleotide modulates the expression of a gene in a plant pathogen.

[0070] Another embodiment of the present invention is the above formulation, wherein the at least one additional active agent is selected from the group consisting of an herbicide, an insecticide, a fungicide, a bactericide, a viricide, and any combination thereof.

[0071] Yet another embodiment of the present invention is a method of regulating expression of a gene in an organism, comprising applying any one of the above formulations to the organism.

[0072] Yet another embodiment of the present invention is a method of modulating a trait of a plant, comprising delivering to the plant an effective amount of the above formulation.

[0073] Another embodiment of the present invention is above method of modulating a trait of a plant, wherein the trait is selected from the group consisting of total seed germination, rate of seed germination, disease tolerance, insect tolerance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, tolerance to heavy metals, total yield, seed yield, fruit yield, root growth, early vigor, plant growth, plant biomass, plant size, plant lifespan, total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number, leaf number, fruit size, fruit freshness, fruit ripening time, fruit nutritional content, plant nutritional content, plant sensitivity to herbicide, and any combination thereof.

[0074] Another embodiment of the present invention is above method of modulating a trait of a plant, wherein one or more of the traits is improved relative to a plant not treated with the formulation.

[0075] Another embodiment of the present invention is above method of modulating a trait of a plant, wherein at least one trait selected from the group consisting of plant growth, plant lifespan, plant size, fruit size, fruit yield, total yield, fruit freshness, fruit ripening time, plant nutritional content, and fruit nutritional content, is improved relative to a plant not treated with the formulation.

[0076] Another embodiment of the present invention is above method of modulating a trait of a plant, wherein one or more of the traits is decreased relative to a plant not treated with the formulation.

[0077] Another embodiment of the present invention is above method of modulating a trait of a plant, wherein the plant growth and/or the plant lifespan is decreased relative to a plant not treated with the formulation.

[0078] Another embodiment of the present invention is above method of modulating a trait of a plant, wherein the fruit ripening time is decreased relative to a plant not treated with the formulation.

[0079] Another embodiment of the present invention is above method of modulating a trait of a plant, wherein the plant sensitivity to herbicide is increased relative to a plant not treated with the formulation.

[0080] Yet another embodiment of the present invention is a method of modulating a trait of an insect, comprising delivering an effective amount of the above formulation to the insect, to a plant infested with the insect, or to a plant prior to infestation with the insect.

[0081] Another embodiment of the present invention is the above method of modulating a trait of an insect, wherein the trait modulated is insect growth,

development,and/or lifespan.

[0082] Yet another embodiment of the present invention is method of modulating the pathogenicity of a plant pathogen, comprising applying the above formulation of to the plant pathogen, to a plant infected with the plant pathogen, or to a plant prior to infection with the plant pathogen.

[0083] Yet another embodiment of the present invention plant cell, insect cell, fungal cell, bacterial cell, or mammalian cell comprising the above compound. [0084] Yet another embodiment of the present invention is a method for screening capped polymer mixtures comprising:

(1) reacting a polymer comprising at least one primary and/or secondary amino group with at least one cap precursor in at least two different ratios based on the total number of nitrogen atoms in the polymer to form at least two different mixtures of capped polymers;

(2) separately combining each of the at least two different mixtures of capped polymers with an oligo- or polynucleotide to form at least two different mixtures of oligo- or

polynucleotide/capped polymer complexes;

(3) separately assaying each of the at least two different mixtures of oligo- or

polynucleotide/capped polymer complexes to determine the ability of each mixture to transfect a cell with the oligo- or polynucleotide.

[0085] Another embodiment of the present invention is the above method for screening capped polymer mixtures, further comprising:

(4) selecting an oligo- or polynucleotide/capped polymer complex mixture determined to be able to transfect a cell with the oligo- or polynucleotide;

(5) separating the mixture into at least two oligo- or polynucleotide/capped polymer complex sub-mixtures on the basis of average molecular weight;

(6) separately assaying each of the at least two different sub-mixtures of to determine the ability of each sub-mixture to transfect a cell with the oligo- or polynucleotide.

[0086] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the polymer and the at least one cap precursor are reacted in three different ratios to form three different mixtures of capped polymers.

[0087] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the three different polymer/cap precursor ratios are 3:3, 3:2, and 3: 1.

[0088] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the oligo- or polynucleotide reduces the expression of a target gene expressed by the cell. [0089] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the assaying comprises measuring expression levels of the target gene.

[0090] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the assaying comprises measuring mRNA levels of the target gene.

[0091] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the mRNA levels are measured by a method selected from the group consisting of quantitative PCR and Northern blot analysis.

[0092] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the assaying comprises measuring expression and/or activity of a protein encoded by the target gene.

[0093] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the expression of the protein is measured by a method selected from the group consisting of Western blot analysis, flow cytometry, ELIS A and mass spectrometry.

[0094] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the fluorescence and/or enzyme activity of the protein is measured.

[0095] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the target gene is endogenous to the cell.

[0096] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the target gene is heterologous to the cell.

[0097] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the target gene encodes a marker protein.

[0098] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the marker protein is Firefly luciferase. [0099] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the marker protein is Green Fluorescent Protein (GFP).

[0100] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the cell also expresses Renilla luciferase.

[0101] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the assaying comprises determining a phenotype of the cell, or an organism comprising the cell.

[0102] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the phenotype comprises changes in growth of the organism.

[0103] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the oligo- or polynucleotide is an siRNA or an antisense RNA.

[0104] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the polymer comprising at least one primary and/or secondary amino group is a polyethyleneimine prepared by the ring-opening polymerization of aziridine, a polyethyleneimine derived from the partial or complete hydrolysis of a poly(2-alkyl-2-oxazoline), or combinations thereof.

[0105] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the poly(2-alkyl-2-oxazoline) is poly(2-ethyl-2- oxazoline).

[0106] Another embodiment of the present invention is the above method for screening capped polymer mixtures, wherein the at least one cap precursor is a compound of (Ilia):

6 (Ilia) wherein

Rs is hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted hetero aliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group; and

R is an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted hetero aliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group; a compound of formula (IVa):

wherein

are, independently, hydrogen or an optionally substituted aliphatic group; R9 is an optionally substituted aliphatic group; and X is O or N; or any combination thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0107] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

[0108] In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Any ranges described herein will be understood to include the endpoints and all values between the endpoints.

[0109] The novel modified PEIs of the present disclosure provide for several different advantages in the delivery of active agents to target organisms. For example, these modified PEIs may be used to complex oligonucleotides and polynucleotides, thereby enhancing their delivery to the target organism and preventing their degradation. These modified PEIs may also be used generate formulations, such as microparticles, nanoparticles, picoparticles, liposomes, and micelles, containing the active agent to be delivered, and which may be done so with improved batch consistency. These modified PEIs, as well as the formulations thereof, may be biocompatible and biodegradable and may be used to provide controlled, sustained release of the active agent to be delivered. These modified PEIs may also act as proton sponges in the delivery of an active agent to a cell to cause endosome lysis. These modified PEIs may also exhibit improved aqueous solubility.

[0110] In one aspect, the present disclosure provides for novel modified

polyethyleneimines comprising a structural unit of formula (I) and/or formula (II):

wherein:

Ri is hydrogen, a group of formula (III):

wherein

P5 is hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted hetero aliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group; and

wherein

X is O or NR11, wherein Rn is selected from the group consisting of H and Ci to

C 10 alkyl groups; or a group of formula (V):

wherein

Rio is selected from the group consisting of Ci to C₄ alkyl groups;

are, independently, hydrogen, a group of formula (III):

wherein R₅ and R₆ are as defined above; or a group of formula (IV):

wherein R₇, Rg, R9, and X are as defined above; wherein the nitrogen atom bonded to Ri in formula (I) and the nitrogen atom bonded to R₄ in formula (II) can each, independently, instead be bonded to an ethylene moiety in the modified polyethyleneimine; and with the proviso that at least one of Ri, R₂, R₃, and R₄ is a group of formula (III) or a group of formula (IV).

[0111] In certain embodiments, each of groups R₂, R₃, and R₄, to the extent that one or more of each is present in the presently disclosed modified polyethyleneimines, can independently be any combination of hydrogen, a group of formula (III), or a group of formula (IV), while each group Ri, to the extent that one or more is present in the presently disclosed modified polyethyleneimines, can independently be any combination of hydrogen, a group of formula (III), a group of formula (IV), or a group of formula (V). Any one of groups Ri, R₂, R₃, and R₄, to the extent that one or more of each is present in the presently disclosed modified polyethyleneimines, can be identical or different to the others. In certain embodiments, at least two of, or at least three of, or all groups Ri, R₂, R₃, and R₄, to the extent that two or more of each is present in the presently disclosed modified

polyethyleneimines, can be identical to each other. In certain embodiments, at least two of, or at least three of, or all groups Ri, R₂, R₃, and R₄, to the extent that two or more of each is present in the presently disclosed modified polyethyleneimines, can be different to each other.

[0112] In certain embodiments, group Ri of the presently disclosed modified polyethyleneimines is a group of formulae (III), (IV), or (V), groups R₂ and/or R₃ of the presently disclosed modified polyethyleneimines is a group of formulae (III) or (IV), and group R₄ of the presently disclosed modified polyethyleneimines is a group of formulae (III) or (IV). In certain embodiments, groups Ri and R^of the presently disclosed modified polyethyleneimines are each a group of formulae (III) or (IV), and groups R₂ and/or R₃ of the presently disclosed modified polyethyleneimines are each a group of formulae (III) or (IV). In certain of these embodiments, Ri and R₄ are each a group of formulae (III) and R₂ and/or R₃ is a group of formulae (III), while in certain other of these embodiments, Ri and R₄ are each a group of formulae (IV) and R₂ and/or R₃ is a group of formulae (IV).

[0113] In certain embodiments, each of groups Ri and R₄ of the presently disclosed modified polyethyleneimines are independently selected from at least two different groups of formulae (III) and/or (IV), and each of groups R₂ and/or R₃ of the presently disclosed modified polyethyleneimines are independently selected from at least two different groups of formulae (III) and/or (IV). In certain of these embodiments, each of groups Ri and R₄ is independently selected from at least two different groups of formulae (III), and each of groups R₂ and/or R₃ is independently selected from at least two different groups of formulae (III), while in certain other of these embodiments, each of groups Ri and R₄ is independently selected from at least two different groups of formulae (IV), and each of groups R₂ and/or R₃ is independently selected from at least two different groups of formulae (IV).

[0114] In certain embodiments, at least one of Ri, R₂, R₃, and R₄ of the presently disclosed modified polyethyleneimines is a group of formula (III). In certain embodiments, at least Ri and R₂ of the presently disclosed modified polyethyleneimines are each, independently, a group of formula (III). In certain embodiments, at least Ri, R₂, and R₃ of the presently disclosed modified polyethyleneimines are each, independently, a group of formula (III). In certain embodiments, Ri, R₂, R₃, and R₄ of the presently disclosed modified polyethyleneimines are each, independently, a group of formula (III). In each of the foregoing embodiments of this paragraph, each of the groups R₅ of the groups of formula (III) may be identical or different to each other and each of the groups R₆ of the groups of formula (III) may be identical or different to each other. In certain of these embodiments, R₅ is hydrogen and R₆ is a Ci to C₂₀ alkyl group. In certain of these embodiments, R₅ is hydrogen and R₆ is a C₄ to C₁₆ alkyl group. In certain of these embodiments, R₅ is hydrogen and R₆ is a C₆ to C₁₄ alkyl group.

[0115] In certain embodiments, where at least Ri and R₂ are each, independently, a group of formula (III), R5 is H and R₆ is selected from the group consisting of groups of formulae (XI) through (XXVII):

(XXIII)

[0116] In certain embodiments, at least one of Ri, R₂, R₃, and R₄ of the presently disclosed modified polyethyleneimines is a group of formula (IV). In certain embodiments, at least Ri and R₂ of the presently disclosed modified polyethyleneimines are each, independently, a group of formula (IV). In certain embodiments, at least Ri, R₂, and R₃ of the presently disclosed modified polyethyleneimines are each, independently, a group of formula (IV). In certain embodiments, Ri, R₂, R₃, and R₄ of the presently disclosed modified polyethyleneimines are each, independently, a group of formula (IV). In each of the foregoing embodiments of this paragraph, each of the groups R₇ of the groups of formula (IV) may be identical or different to each other, each of the groups Rg of the groups of formula (IV) may be identical or different to each other, each of the groups R₉ of the groups of formula (IV) may be identical or different to each other, and each X of the groups of formula (IV) may be identical or different to each other. In certain of these embodiments, R₇ and Rg is hydrogen, R₉ is a Ci to C₂o alkyl group, and X is O or NH. In certain of these

embodiments, R₇ and Rg is hydrogen, R₉ is a Cg to Ci₆ alkyl group, and X is O or NH.

[0117] In certain embodiments, where at least Ri and R₂ are each, independently, a group of formula (IV), R₇ and Rg are each hydrogen,_, X is O, and R₉ is a group selected from the group consisting of formulae (XXVIII), (XXIX), and (XXX):

and (XX

X).

In certain other embodiments, where at least Ri and R₂ are each, independently, a group of formula (IV), R₇ and Rg are each hydrogen,_, X is NH, and R9 is a group selected from the group consisting of formulae (XXIX) and (XXX):

X).

[0118] In certain embodiments, the presently disclosed modified polyethyleneimines can be branched or linear. In certain embodiments, the branched modified polyethyleneimine can be hyperbranched or dendritic. The branched or linear modified polyethyleneimine can be derived from a branched or linear polyethyleneimine of any suitable number average molecular weight (M_n). In certain embodiments, the branched modified polyethyleneimine of the present disclosure is derived from a branched polyethyleneimine having a number average molecular weight in the range of from 600 to 10,000 Daltons. In certain other embodiments, the branched modified polyethyleneimine of the present disclosure is derived from a branched polyethyleneimine having a number average molecular weight of greater than 2,000 Daltons. In certain embodiments, the branched modified polyethyleneimine of the present disclosure is derived from a branched polyethyleneimine having a number average molecular weight selected from the group consisting of 600, 1,200, 1,800, and 10,000 Daltons. In certain embodiments, the linear modified polyethyleneimine of the present disclosure is derived from a linear polyethyleneimine having a number average molecular weight in the range of from 2,500 to 100,000 Daltons. In certain other embodiments, the linear modified polyethyleneimine of the present disclosure is derived from a linear polyethyleneimine having a number average molecular weight of greater than 2,000 Daltons. In certain embodiments, the linear modified polyethyleneimine of the present disclosure is derived from a linear polyethyleneimine having a number average molecular weight of 2,500 Daltons. [0119] In the group of formula (III), R₅ and R₆ can, independently, each be an optionally substituted aliphatic or eye lo aliphatic group. In the group of formula (IV), R₇, Rg, and R9 can, independently, each be optionally substituted aliphatic group. The aliphatic groups of the presently disclosed modified PEIs refer to both saturated and unsaturated aliphatic hydrocarbyl groups, which can be straight chain (i.e. , unbranched), branched, or cyclic (including polycyclic) and are optionally substituted with one or more functional groups. Examples of aliphatic groups include, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl groups, each of which are optionally substituted with one or more functional groups. In the group of formula (IV), Rn can be a Ci to C₁₀ alkyl group, while in the group of formula (V), Rio is a Ci to C₄ alkyl group. As used herein, the term "alkyl" refers to saturated hydrocarbyl groups, which can be unbranched, branched, or cyclic (i.e. , alicyclic) alkyl groups. As used herein, the terms "alkenyl" and "alkylene" refers to unsaturated hydrocarbyl groups having at least one carbon-carbon double bond. As used herein, the term "alkynyl" refers to unsaturated hydrocarbyl groups having at least one carbon-carbon triple bond.

[0120] Examples of such aliphatic groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH₂-cyclopropyl, n-butyl, sec-butyl, isobutyl, tert- butyl, cyclobutyl, -CH₂-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, neopentyl, cyclopentyl, -CH₂-cyclopentyl, hexyl, cyclohexyl, -CH₂-cyclohexyl, heptyl, cycloheptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icosenyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tridecynyl,

tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, octadecynyl, nonadecynyl, icosynyl, and all isomers thereof.

[0121] In the group of formula (III), R₅ and R₆ can, independently, each be an optionally substituted hetero aliphatic or heterocycloaliphatic group. The hetero aliphatic groups of the presently disclosed modified PEIs refers to aliphatic groups, as described above, that independently in one or more instances contain an oxygen, sulfur, nitrogen, phosphorus, or silicon atom between two carbon atoms of the aliphatic group. Such hetero aliphatic groups include saturated and unsaturated heterocycles. As used herein, the term "heterocycles," refers to a non-aromatic partially unsaturated or fully saturated 3- to 10- membered ring system, which includes single rings of 3 to 8 atoms in size and bi- and tricyclic ring systems, which may include aromatic six-membered aryl or aromatic heterocyclic groups fused to a non-aromatic heterocyclic ring. Such heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. Examples of such heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,

isothiazolidinyl, 2,5-dihydro- lH- imidazole, and tetrahydrofuryl.

[0122] In certain embodiments, the aliphatic and/or heteroaliphatic groups of the modified PEIs of the present disclosure independently contain from 1 to 20, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 5, or from 1 to 4, or from 4 to 5, or from 4 to 8, or from 4 to 10, or from 4 to 12, or from 4 to 20, or from 5 to 20, or from 5 to 12, or from 5 to 10, or from 5 to 8, or from 8 to 10, or from 8 to 12, or from 8 to 20, or from 10 to 12, or from 10 to 20, or from 12 to 20 carbon atoms.

[0123] In the group of formula (III), R₅ and R₆ can, independently, each be an optionally substituted aralkyl or aryl group. The aryl and heteroaryl groups of the presently disclosed modified PEIs refer to mono- or polycyclic aromatic carbocyclic groups and mono- or polycyclic aromatic heterocyclic groups. In certain embodiments, these groups have 3- 14 carbon atoms, each of which is optionally substituted. In certain embodiments, the aryl group is a mono- or bicyclic carbocyclic ring system having one or two aromatic rings. The term "carbocyclic" as used herein, refers to an aromatic or non-aromatic ring system in which each atom of the ring is a carbon atom. Examples of such aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, and indenyl. In certain embodiments, the heteroaryl group is a mono- or bicyclic heterocyclic ring system having one or two aromatic rings. In certain embodiments, the heteroaryl group (1) has from five to ten ring atoms of which one ring atom is selected from S, O, and N, (2) has zero, one, or two ring atoms that are additional heteroatoms independently selected from S, O, and N, and (3) the remaining ring atoms are carbon. Examples of such heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, and isoquinolinyl. [0124] It will be appreciated that the compounds of the presented disclosure may be substituted with any number of substituents. In general, the term "substituted," whether preceded by the term "optionally" or not, and substituents contained in formulas of the present disclosure, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. Broadly, permissible substituents include all acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For purposes of this disclosure, heteroatoms, such as nitrogen, may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, the presently disclosed modified PEIs are not intended to be limited in any manner by the permissible substituents of organic compounds. Any substituents disclosed herein may also be further substituted (e.g. , an aryl substituent may itself be substituted, such as with another aryl group, which, in turn, is further substituted with fluorine at one or more positions).

[0125] Examples of optional substituents of the modified PEIs of the present disclosure include, but are not limited to, aliphatic groups, hetero aliphatic groups, aryl groups, heteroaryl groups, arylalkyl groups, heteroarylalkyl groups, alkoxy groups, aryloxy groups, heteroalkoxy groups, heteroaryloxy groups, alkylthio groups, arylthio groups, hetero alky lthio groups, hetero arylthio groups, F, CI, Br, I, -OH, -N0₂, -CN, -CF₃, -CH₂CF₃, - CHC1₂, -CH₂OH, -CH₂CH₂OH, -CH₂NH₂, -CH₂S0₂R, -C(0)R, -C0₂(R), -CON(R)₂, - OC(0)R, -OC0₂R, -OCON(R)₂, -N(R)₂, -S(0)₂R, and -NR(CO)R, wherein each R is, independently, hydrogen, an aliphatic group, a hetero aliphatic group, an aryl group, a heteroaryl group, an arylalkyl group, or a heteroarylalkyl group, wherein any of the aliphatic, hetero aliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be optionally substituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be optionally substituted.

[0126] In certain embodiments, the presently disclosed modified PEIs can be a salt, either from protonation of one or more primary, secondary, and/or tertiary amino groups of the modified PEIs by a mineral or organic acid or by quaternization of one more available tertiary amino groups of the modified PEIs. Examples of counterions for such salts include, but are not limited to, halides, such as fluoride, chloride, bromide, or iodide, nitrate, hydrogen sulfate, dihydrogen phosphate, bicarbonate, nitrite, perchlorate, iodate, chlorate, bromate, chlorite, hypochlorite, hypobromite, cyanide, amide, cyanate, hydroxide, permanganate, an acid anion such as acetate or formate, or anions with negative charges greater than - 1 (e.g., having in some embodiments one or more than one adsorbent functional group as counterion), such as oxide, sulfide, nitride, arsenate, phosphate, arsenite, hydrogen phosphate, sulfate, thio sulfate, sulfite, carbonate, chromate, dichromate, peroxide, or oxalate.

[0127] In certain embodiments, the presently disclosed modified PEIs, and optional substitutents thereon, can contain isotopes of various common atoms. Examples of such

13 15 18 18

isotopes include, but are not limited to, deuterium, C , N , O , and F . In certain embodiments, modified PEIs, independently, can be perfluorinated C_1-2o alkyl groups.

[0128] In certain embodiments, the presently disclosed modified PEI is selected from the group consisting of compounds of formulae (1) through (299):

the number average molecular weight of the unmodified

polyethyleneimine prior to modification;

L/B denotes whether the modified polyethyleneimine is linear or branched;

[0129] In certain embodiments, the presently disclosed modified PEIs are relatively no n-cyto toxic. In certain embodiments, the presently disclosed modified PEIs are biocompatible and biodegradable. In certain embodiments, the presently disclosed modified PEIs have a pKa in the range of from about 3.0 to about 9.0, or in the range of from about 5.0 to about 8.0, or in the range of from about 5.5 to about 7.5, or in the range of from about 6.0 to about 7.0.

[0130] The presently disclosed modified PEIs may exist in particular geometric or stereoisomeric forms. The present disclosure contemplates all such forms, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent, such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this disclosure.

[0131] Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present disclosure. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90: 10, 95:5, 96:4, 97:3, 98:2, 99: 1, or 100:0 isomer ratios are all contemplated by the present disclosure. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures. [0132] If a particular enantiomer of the presently disclosed modified PEIs is desired, it may be prepared by asymmetric synthesis, for example, by reacting an unmodified or partially modified polyethyleneimine with a chiral epoxide, acrylate, or acrylamide. It may also be prepared by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts can be formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

[0133] In certain embodiments, where the presently disclosed modified PEI contains at least one group of formula (III), the modified PEI can contain at least one stereocenter. Therefore, in certain embodiments of the presently disclosed modified PEIs, each group of formula (III):

is, independently, a group of formulae (Ilia) or (Illb):

[0134] The "enantiomeric excess" of a substance is a measure of how pure a desired enantiomer is relative to the undesired enantiomer. Enantiomeric excess is defined as the absolute difference between the mole fraction of each enantiomer which is most often expressed as a percent enantiomeric excess. For mixtures of diastereomers, there are analogous definitions and uses for "diastereomeric excess" and percent diastereomeric excess. For example, a sample with 70% of R isomer and 30% of S will have an enantiomeric excess of 40%. This can also be thought of as a mixture of 40% pure R with 60% of a racemic mixture (which contributes 30% R and 30% S to the overall composition).

[0135] The modified PEIs of the present disclosure can have an enantiomeric excess or a diastereomeric excess up to and including 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%.

[0136] The presently disclosed modified PEIs may be prepared by any method known in the art. In certain embodiments, the modified PEIs of the present disclosure are synthesized by reacting an unmodified polyethyleneimine, a partially modified

polyethyleneimine, or a partially hydrolyzed poly(2-alkyl-2-oxazoline) with one or more terminal epoxides, one or more interior epoxides, one or more acrylates, one or more acrylamides, or any combination thereof. In certain embodiments, these starting materials are commercially available and/or are easily and/or inexpensively prepared. Scheme 1 depicts these reactions where a linear or branched PEI is reacted with a terminal epoxide, an acrylate, or an acrylamide:

Scheme 1

[0137] In certain embodiments, the unmodified polyethyleneimine, partially modified polyethyleneimine, partially hydrolyzed poly(2-alkyl-2-oxazoline) is reacted in the presence of a stoichiometric amount or stoichiometric excess (based on the theoretical stoichiometry of available amino groups in the polymer) of a terminal epoxide, interior epoxide, acrylate, acrylamide, or any combination thereof. In certain embodiments, the synthesis of the presently disclosed modified PEIs is performed without solvent (i.e., neat). In certain other embodiments, the synthesis of the presently disclosed modified PEIs is performed in a suitable solvent, such as a lower alcohol. In certain embodiments, these syntheses are performed at a temperature in the range of about 90 to about 120 °C for about 2 to about 96 hours. In certain embodiments, conventional heating sources can be employed. In certain other embodiments, non-conventional heating sources, such as microwave radiation, can be employed. After the reaction is complete, the reaction mixture is cooled, any solvent used is removed in vacuo, and the crude product is purified or used "as us." .

[0138] The branched modified polyethyleneimine of the present disclosure can be derived from a branched polyethyleneimine comprising primary, secondary, and tertiary amino groups in any suitable ratio. In certain embodiments, the branched modified polyethyleneimine of the present disclosure can be derived from a branched

polyethyleneimine comprising primary, secondary, and tertiary amino groups present in a ratio of 1:2: 1.

[0139] In certain embodiments, the modified polyethyleneimine of the present disclosure is derived from the partial hydrolysis of a functionalized poly(2-oxazoline)s, such as a poly(2-alkyl-2-oxazoline) or a poly(2-aryl-2-oxazoline). Examples of such poly(2-alkyl- 2-oxazoline)s include, but are not limited to, poly(2-methyl-2-oxazoline), poly(2-ethyl-2- oxazoline), poly(2-propyl-2-oxazoline), poly(2-butyl-2-oxazoline), poly(2-pentyl-2- oxazoline), poly(2-hexyl-2-oxazoline), poly(2-heptyl-2-oxazoline), poly(2-octyl-2- oxazoline), poly(2-nonyl-2-oxazoline), poly(2-decyl-2-oxazoline), and poly(2-undecyl-2- oxazoline). An example of such a poly(2-aryl-2-oxazoline) includes, but is not limited to, poly(2-phenyl-2-oxazoline). These and other examples of poly(2-alkyl-2-oxazoline)s and poly(2-aryl-2-oxazoline)s, as well as other examples of functionalized poly(2-oxazoline)s, are disclosed in "Design Strategies for Functionalized Poly(2-oxazolines)s and Derived

Materials," Rossegger et al., Polymers, Vol. 5, pages 956-1011 (2013), which is incorporated herein by reference in its entirety. These functionalized poly(2-oxazoline)s can be further functionalized at the termination of polymerization through the use of various terminating agents, which become end groups on the resulting functionalized poly(2-oxazoline). An example of such a terminating agent includes, but is not limited to, morpholine. Additional examples of terminating agents are disclosed in "Design Strategies for Functionalized Poly(2- oxazolines)s and Derived Materials," Rossegger et al., Polymers, Vol. 5, pages 956-1011 (2013), which is incorporated herein by reference in its entirety. In certain embodiments, the average molecular weight of the poly(2-alkyl-2-oxazoline) is in the range of from about 2,000 Daltons to aboute 5,000 Daltons. In certain embodiments, hydrolysis of the poly(2-alkyl-2- oxazoline) can be achieved and controlled by treatment with concencetrated acid (e.g., HC1) over defined time periods. In certain embodiments, such treatment results in a hydrolysis percentage of 5, 10, 12.5, 15, 17.5, 20, 22.5, 25, and 50 %. Partially hydrolyzed poly(2-alkyl- 2-oxazoline) is structurally similar to partially protected PEI, but with narrower

polydispersity indices.

[0140] The synthesized modified PEIs of the present disclosure may be purified by any technique known in the art including, but not limited to, precipitation, crystallization, silica gel chromatography, size exclusion chromatography, ion-exchange chromatography, HPLC, and distillation.

[0141] In certain embodiments, modified PEIs of the present disclosure can be synthesized such that the modified PEI contains two or more different groups of Formula (III), two or more different groups of Formula (IV), or one or more groups of both Formulae (III) and (IV). In certain embodiments, this can be achieved by reacting the unmodified PEI or partially hydrolyzed poly(2-alkyl-2-oxazoline) with a less-than-stoichiometric amount of a terminal epoxide, interior epoxide, acrylate, or acrylamide, followed by reacting the now- partially modified PEI with a further, different terminal epoxide, interior epoxide, acrylate, or acrylamide, and so on. In certain other embodiments, this can be achived by reacting the unmodified PEI or partially hydrolyzed poly(2-alkyl-2-oxazoline) with a mixture of different terminal and/or interior epoxides or a mixture of different acrylates and/or acrylamides. In certain embodiments this can be achieved through substituting one or more of the amino groups of the unmodified PEI, partially modified PEI, or partially hydrolyzed poly(2-alkyl-2- oxazoline) with a protecting group and then reacting the remaining unprotected amino groups of the unmodified PEI, partially modified PEI, or partially hydrolyzed poly(2-alkyl-2- oxazoline) with a terminal epoxide, interior epoxide, acrylate, or acrylamide. The protecting groups can then be removed and the now unprotected amino groups of the partially modified PEI can then be further reacted with a different terminal epoxide, interior epoxide, acrylate, or acrylamide.

[0142] Examples of amino -protecting groups thai may be used include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2- sulfojfluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl- [9-( 10, 10-dioxo- 10, 10, 10, 10-tetrahydromioxanthyl) ] methyl carbamate (DBD-Tmoc), 4- methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2- trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), l-(l-adamantyl)-l- methylethyl carbamate (Adpoc), 1,1 -dimethyl- 2-haloethyl carbamate, lJ-dimethyl-2,2- dibromoethyl carbamate (DB-t-BOC), l,l-dimethyl-2,2,2-trichloroethyl carbamate

(TCBOC), l-memyI-l-(4-biphenylyl)etbyl carbamate (Bpoc), l-(3,5-di-t-butylpherjyl)-l- methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N- dicyclohex.ylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), ally! carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N- hydroxypiperidinyi carbamate, alkyklithio carbamate, benzyl carbamate (Cbz), p- methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p- chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate ( Msz ). 9-anthryImethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyI carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-( 1 ,3- dithianyl)] methyl carbamate (Dmoc), 4 -methylthio phenyl carbamate (Mtpc), 2,4- dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2- triphenylphosphonioisopropyl. carbamate (Ppoc), l,l -dimethyl-2-cyanoethyl carbamate, m- chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5- benzisoxazo lylmethy 1 carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4- dimethoxy-6-nitrobenzyl carbamate, phenyl(o-rjitrophenyl)methyl carbamate, phenothiazinyl- (lO)-carbonyl derivative, N'-p-toluenesulfonylaminocarbonyl derivative, N'- phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl thiocarbamate, p- cyanobenzyl carbamate, cyclobutyl carbamate, cvclohexyl carbamate, cyclopentvl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzy 1 carbamate, 1 , 1 -dimethyl-3-(N,N- dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2- pyridylsmethyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, l-rnethylcyelohexyl carbamate, 1 -methyl- 1- cyclopropylmethyl carbamate, 1 -methyl- l-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl- 1 -(p-phenylazophenyl)ethyl carbamate, 1 -methyl- 1 -pheny lethyl carbamate, 1 -methyl- 1-(4- pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t- butylphenyl carbamate, 4-(trimethylammonium) benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, aceiamide, chioroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N- benzoy! pheny !alanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o- nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p- hydroxyphenyljpropanamide, 3-(o-nitrophenyl)propa.namide, 2-methyl-2-(o- nitrophenoxy)propanamide, 2- methyl- 2-(o-phenylaz.ophenoxy)propanamide, 4- chlorobutanamide, 3-met.hyl-3-nitrobutan.amide, o-nitrocinnamide, N-a.cetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2- one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenyimaleimide, N-2,5- dimethylpyrrole, Nf- 1 , 1 ,4,4-tetramethyldisily lazacyclopentane adduct (STABASE), 5- substituted 1 ,3-dimethyl- 1 ,3 ,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl- 1,3,5- t.riaza.cyclohexan-2-one, 1 -substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N- [2-(trimethylsilyl)ethoxy] methylamine (SEM), N-3-acetoxypropylamine, N-(l-isopropyl- 4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di{4- methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N- [(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N- 2,7-dichloro-9-f3.uorenyl.methyleneamine, N-ferrocenylmethylamino (Fcm), N-2- picolylamino N'-oxide, N-l,l-dimethylthiomethyleneamine, N-benzyiideneamine, N-p- xnethoxybenzylideneamme, N-diphenylmethyleneamme, N-[(2- pyridyl)mesityl]methyleneamine, N (N',N'-dimethylaminomethylene)amine, Ν,Ν'- isopropylidenediamine, N-p-nitrobenzylidenearmiie, N-salicylideneamine, N-5- chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N- cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-l-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentacarbonylchromium- or

tungsten)carbonyl] amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphmamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzene sulfanamide, o-nitrobenzenesulfenamide (Nps), 2,4- dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4- methoxybenzenesulfenarmde, triphenylmethylsulfenamide, 3-nitropyridmesulfenarmde (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4- methoxybenzenesulfonamide (Mir), 2,4,6-trimethoxybeiizenesulfonainide (Mtb), 2,6- dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4- methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonaxnide (iMcls), 2,2,5,7,8-pentamethylchroman-6-siilfonamide (Pmc), methanesulfonamide (Ms), β- trimethylsilylethane sulfonamide (SES), 9-antbracenesulfonamide, 4-(4',8'- dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide,

trifluoromethylsulfonamide, and phenacylsulfonamide.

Modified PEI-Based Formulations

[0143] In another aspect, the present disclosure provides for formulations comprising the presently disclosed modified PEIs that may be used to deliver one or more active agents to a target organism. Therefore, in certain embodiments, the presently disclosed formulations can comprise at least one modified PEI of the present disclosure and at least one active agent to be delivered. In certain embodiments, the at least one modified PEI of the present disclosure and a first active agent to be delivered are non-covalently associated to one another to form a non-covalent complex. As used herein, the term "non-covalently associated" encompasses any kind of intermolecular interaction between the at least one modified PEI of the present disclosure and the first active agent to be delivered other than covalent interactions (i.e., interactions that involve the sharing of electrons). Examples of such non- covalent interactions include, but are not limited to, electrostatic interactions, such as ionic interactions, hydrogen bonding, and halogen bonding, Van der Waals forces, such as the Keesom force, the Debye force, and London dispersion forces, π-effects, such as π-π interactions, cation-π interactions, anion-π interactions, and polar π interactions, and hydrophobic interactions. As such, the term "non-covalent complex," as used herein, encompasses a complex of at least one modified PEI of the present disclosure and a first active agent to be delivered wherein the least one modified PEI and the first active agent are associated to each other via non-covalent interactions, as defined above. [0144] In certain embodiments, the presently disclosed formulations further comprise at least one additional active agent to be delivered. In certain embodiments, this at least one additional active agent is part of the non-covalent complex of the least one modified PEI and the first active agent. In other words, the at least one additional active agent can be contained within the non-covalent complex or adhered to the surface of the non-covalent complex via non-covalent interactions, as defined above. In certain other embodiments, the at least one additional active agent is not contained within the non-covalent complex or adhered to the surface of the non-covalent complex, e.g. , the at least one additional active agent is simply in a physical mixture with the non-covalent complex. In certain embodiments, the first active agent is an oligonucleotide or a polynucleotide, and the at least one additional active agent is an herbicide, an insecticide, a fungicide, a bactericide, and/or a viricide. In certain embodiments, the first active agent is used to increase the sensitivity of the target organism to the additional active agent, for example, to increase the sensitivity of a plant to an herbicide, or to increase the sensitivity of an insect to an insecticide.

[0145] The presently disclosed formulations may also comprise one or more excipients. Suitable excipients include, but are not limited to, fillers, extenders, binders, humectants, disintegrants, plasticizers, stabilizers, solution retarding agents, wetting agents, suspending agents, thickening agents, absorbents, lubricants, surfactants, buffering agents, diluents, solvents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, opacifying agents, separating agents, and coating permeability adjusters. The one or more excipients may be selected from the group consisting of sterols, carbohydrates, proteins, lipids, water-soluble polymers, and any combination thereof. In certain embodiments, the one or more excipients is a phytosterol. In certain other embodiments, the one or more excipients is cholesterol. In certain other embodiments, the one or more excipients comprises a water-soluble polymer such as polyethylene glycol (PEG), a polypropylene oxide (PPO), a polyvinylpyrrolidone (PVP), a polyvinyl alcohol (PVA), a polylactic acid (PLA), a poly(lactic-co-glycolic acid) (PLGA), or any combination thereof. In certain embodiments, the water-soluble polymer can be contained within or adhered to the surface of the non-covalent complexes of the present disclosure via non- covalent interactions, as defined above. In certain other embodiments, the water-soluble polymer can be tethered to the surface of the non-covalent complexes of the present disclosure via a lipid tail that is covalently bound on one end to the water-soluble polymer and which is entrained within the surface and/or interior of the non-covalent complex. [0146] The presently disclosed modified PEIs, being cationic in nature, are capable of encapsulating naked RNA by electrostatic interaction with the phosphate backbone of the RNA to form polyplexes. Therefore, in certain embodiments, the presently disclosed modified PEIs can be used as efficient transfection/delivery vectors. In certain embodiments, various sizes of polyplexes can be formed by varying the nitrogen to phosphate charge ratio. In certain embodiments, these polyplexes can be further stabilized by addition of PEG, which also reduces in vivo toxicity.

[0147] In certain embodiments, the presently disclosed formulations are combined with an agriculturally acceptable carrier. The agriculturally acceptable carrier can be solid or liquid and is a substance useful in formulation of agricultural products. Examples of such agricultural products include, but are not limited to, fertilizers, herbicides, insecticides, fungicides, bactericides, viricides, and nematicides. Examples of such agriculturally acceptable carriers for use in the presently disclosed formulations include, but are not limited to, surface active agents, stickers, spreader stickers, inert carriers, preservatives, humectants, dyes, UV (ultra-violet) protectants, buffers, flow agents, antifoams (e.g. ,

polydimethylsiloxane), sodium alumino silicate, or other components which facilitate product handling and application of the compositions. Examples of agriculturally acceptable inert carriers include inorganic minerals, such as kaolin, mica, gypsum, fertilizer, carbonates, sulfates, and phosphates, organic materials, such as sugar, starches, and cyclodextrins, and botanical materials, such as wood products, cork, powdered corn cobs, rice hulls, peanut hulls, and walnut shells. Agriculturally acceptable carriers are described, for example, in U.S. Patent No. 6,984,609. In certain embodiments, the agriculturally acceptable carriers include, for example, natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, thickeners, binders, or fertilizers. Such carriers are described, for example, in WO 97/33890.

[0148] The presently disclosed formulations may further comprise one or more additional compounds to facilitate passage of the active agent(s) through the plant cell wall. Several technologies for facilitating passage of compounds through the plant cell wall are known in the art. For example, U.S. Patent No. 8,609,420 describes conjugation of the active agent to a semi-conductor nanoparticle within the size range of 3-5 nm (e.g. , a "quantum dot") and one or more cell penetrating peptides to improve penetration of the plant cell and intracellular delivery of the active agent. U.S. Patent No. 8,686,222 describes interacting a polyamidoamine dendrimer and one or more cell penetrating peptides with the active agent to improve cell penetration. U.S. Patent No. 8,653,327 describes delivery of active agents through plant cell walls by coating a PEGylated semiconductor nanoparticle with the active agent. U.S. Patent No. 8,722,410 describes transferring active agents into plant cells by applying the active agent to a nanoparticle coated with a subcellular compartment targeting protein. U.S. Patent Nos. 8,609,420, 8,686,222, 8,653,327, and 8,722,410 are incorporated by reference herein in their entireties.

[0149] The formulations of the present disclosure can take any form. Examples of such forms include, but are not limited to, complexes, particles (e.g. , microparticles, nanoparticles, and picoparticles), micelles, and liposomes. In certain embodiments, two or more active agents (e.g. , two or more siRNA) can be formulated with the presently disclosed modified PEIs to form a single complex, particle, micelle, or liposome containing the two or more active agents. Alternatively, in certain embodiments, the two or more active agents can be separately formulated to form a single complex, particle, micelle, or liposome, each containing a single active agent, and are then combined to form a mixture prior to delivery to a target organism.

[0150] The modified PEIs of the present disclosure possess one or more primary, secondary, and/or tertiary amino groups that, although hindered, are available to interact with an active agent (e.g. , a polynucleotide). As such, a non-covalent complex is formed when an active agent is contacted with the presently disclosed modified PEIs under conditions suitable to form an active agent/modified PEI complex. In certain embodiments, multiple modified PEI molecules may non-covalently complex with an active agent molecule. The non- covalent complex may include 1- 100 modified PEI molecules, 1- 1000 modified

polyethyleneimine molecules, 10- 1000 modified PEI molecules, or 100- 10,000 modified PEI molecules.

[0151] The modified PEIs of the present disclosure may be used to encapsulate active agents. The modified PEIs of the present disclosure have several properties that make them particularly suitable in the preparation of drug delivery devices. These include, but are not limited to: (1) the ability of the modified PEI to complex and "protect" labile agents, (2) the ability to buffer the pH in the endosome (3) the ability to act as a "proton sponge" and cause endosomolysis, and (4) the ability to neutralize the charge on negatively charged agents. Thus, in certain embodiments, the modified PEIs of the present disclosure are used to form particles containing at least one active agent to be delivered. These particles may include other materials, such as proteins, carbohydrates, synthetic polymers (e.g., PEG, PLGA), and natural polymers.

[0152] In certain embodiments, the presently disclosed modified PEIs are combined with an active agent to be delivered to a cell or a subject to form microparticles,

nanoparticles, liposomes, or micelles. The agent to be delivered by the particles, liposomes, or micelles may be in the form of a gas, liquid, or solid, and the agent may be a

polynucleotide, protein, peptide, or small molecule. The presently disclosed modified PEIs may be combined with other modified PEIs of the present disclosure, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, and lipids, to form the particles.

[0153] In certain embodiments, the diameter of the particles of the present disclosure range from 1 to 1,000 micrometers. In certain embodiments, the diameter of the particles range from 1 to 100 micrometers. In certain embodiments, the diameter of the particles range from 1 to 10 micrometers. In certain embodiments, the diameter of the particles range from 10 to 100 micrometers. In certain embodiments, the diameter of the particles range from 100 to 1,000 micrometers. In certain embodiments, the diameter of the particles range from 1 to 5 micrometers. In certain embodiments, the diameter of the particles range from 1 to 1,000 nm. In certain embodiments, the diameter of the particles range from 1 to 100 nm. In certain embodiments, the diameter of the particles range from 1 to 10 nm. In certain embodiments, the diameter of the particles range from 10 nm to 100 nm. In certain embodiments, the diameter of the particles range from 100 nm to 1,000 nm. In certain embodiments, the diameters of the particles range from 1 to 5 nm. In certain embodiments, the diameter of the particles range from 1 to 1,000 pm. In certain embodiments, the diameter of the particles range from 1 to 100 pm. In certain embodiments, the diameter of the particles range from 1 to 10 pm. In certain embodiments, the diameter of the particles range from 10 to 100 pm. In certain embodiments, the diameter of the particles range from 100 to 1,000 pm. In certain embodiments, the diameter of the particles range from 1 to 5 pm.

[0154] The particles of the present disclosure may be prepared using any method known in the art. These include, but are not limited to, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, simple and complex coacervation, and other methods well known to those of ordinary skill in the art. In certain embodiments, methods of preparing the particles are the double emulsion process and spray drying. In other embodiments, methods of preparing the particles are nanoprecipitation or flash precipitation, for example, as disclosed in U.S. Patent Nos. 8,207,290, 8,404,799, 8,546,521, 8,618,240, and 8,809,492, each of which are incorporated herein in its entirety. The conditions used in preparing the particles may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology, "stickiness", shape, etc.). The method of preparing the particle and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may also depend on the agent being encapsulated and/or the composition of the matrix. Methods developed for making particles for delivery of encapsulated agents are described in the literature (e.g., Doubrow, M., Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy," CRC Press, Boca Raton, 1992; Mathiowitz and Langer, J. Controlled Release 5: 13-22, 1987; Mathiowitz et al. Reactive Polymers 6:275-283, 1987; Mathiowitz et al. J. Appl. Polymer Sci. 35:755-774, 1988; each of which is incorporated herein by reference in their entirety).

[0155] If the particles prepared by any of the above methods have a size range outside of the desired range, the particles can be sized, for example, using a sieve. The particle may also be coated. In certain embodiments, the particles are coated with a targeting agent. In other embodiments, the particles are coated to achieve desirable surface properties (e.g., a particular charge).

[0156] The presently disclosed modified PEIs may be used to prepare micelles or liposomes containing an active agent to be delivered. Many techniques for preparing micelles and liposomes are known in the art, and any method may be used with the inventive modified PEIs to make micelles and liposomes. Micelles and liposomes are particularly useful in delivering hydrophobic agents, such as hydrophobic small molecules.

[0157] In certain embodiments, liposomes containing modified PEIs of the present disclosure are formed through spontaneous assembly. In other embodiments, these liposomes are formed when thin lipid films or lipid cakes are hydrated and stacks of lipid crystalline bilayers become fluid and swell. The hydrated lipid sheets detach during agitation and self- close to form large, multilamellar vesicles (LMV). This prevents interaction of water with the hydrocarbon core of the bilayers at the edges. Once these particles have formed, reducing the size of the particle can be modified through input of sonic energy (sonication) or mechanical energy (extrusion). See Walde, P. "Preparation of Vesicles (Liposomes)" In Encyclopedia of Nanoscience and Nanotechnology; Nalwa, H. S. Ed. American Scientific Publishers: Los Angeles, 2004; Vol. 9, pp. 43-79; Szoka et al. "Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes)" Ann. Rev. Biophys. Bioeng. 9:467- 508, 1980; each of which is incorporated herein in its entirety.

[0158] The preparation of liposomes of the present disclosure involves preparing the modified PEIs for hydration, hydrating the modified PEIs with agitation, and sizing the vesicles to achieve a homogenous distribution of liposomes. Modified PEIs are first dissolved in an organic solvent to assure a homogeneous mixture. The solvent is then removed to form a lipidoid film. This film is thoroughly dried to remove residual organic solvent by placing the vial or flask on a vacuum pump overnight. Hydration of the lipidoid film/cake is accomplished by adding an aqueous medium to the container of dry lipidoid and agitating the mixture. Disruption of LMV suspensions using sonic energy typically produces small unilamellar vesicles (SUV) with diameters in the range of from 15 to 50 nm. Lipid extrusion is a technique in which a lipid suspension is forced through a polycarbonate filter with a defined pore size to yield particles having a diameter near the pore size of the filter used. Extrusion through filters with 100 nm pores typically yields large, unilamellar vesicles (LUV) with a mean diameter of from 120 to 140 nm.

[0159] Certain modified PEIs of the present disclosure can spontaneously self- assemble around certain molecules, such as DNA and RNA, to form liposomes. In some embodiments, the application is the delivery of polynucleotides to a target cell. Thus, use of the modified PEIs of the present disclosure allows for simple assembly of liposomes without the need for additional steps or devices such as an extruder.

[0160] The complexes, microparticles, nanoparticles, picoparticles, liposomes, and micelles of the present disclosure may be modified to include targeting agents since it is often desirable to target a particular cell, collection of cells, or tissue. A variety of targeting agents that direct pharmaceutical compositions to particular cells are known in the art (e.g., Cotten et al. Methods Enzym. 217:618, 1993; which is incorporated herein by reference in its entirety). The targeting agents may be included throughout the particle or may be only on the surface. The targeting agent may be a protein, peptide, carbohydrate, glycoprotein, lipid, small molecule, and/or nucleic acid. The targeting agent may be used to target specific cells or tissues or may be used to promote endocytosis or phagocytosis of the particle. Examples of targeting agents include, but are not limited to, antibodies, fragments of antibodies, low- density lipoproteins (LDLs), transferrin, asialycoproteins, gpl20 envelope protein of the human immunodeficiency virus (HIV), carbohydrates, receptor ligands, sialic acid, and aptamers. If the targeting agent is included throughout the particle, the targeting agent may be included in the mixture that is used to form the particles. If the targeting agent is only on the surface of the particle, the targeting agent may be associated with (i.e., by covalent, hydrophobic, hydrogen bonding, van der Waals, or other interactions) the formed particles using standard chemical techniques.

[0161] In certain embodiments, the formulations of the present disclosure can be formulated as a bait, a food substance, or an attractant. For example, the formulations of the present disclosure can be incorporated into an insect bait suitable for oral administration of the formulation to the target insect. The bait may comprise a formulation comprising an active agent and a modified PEI of the present disclosure dispersed in a carrier and an edible insect attractant. In certain embodiments, the bait comprises an edible insect attractant and a nanoparticle or microparticle comprising at least one active agent and at least one modified PEI of the present disclosure, wherein the nanoparticle or microparticle is dispersed in a carrier. The formulation of the present disclosure and attractant can be mixed together before being dispersed in the desired carrier. Suitable attractants include any type of insect food and/or attractant which will lure the insect to the bait to ingest the bait. Exemplary insect foods or attractants include, but are not limited to, any type of insect food, including various sugars, proteins, carbohydrates, yeast, fats, and/or oils. The bait can be in any form suitable for delivery and ingestion of the composition, depending on the habitat and target insect, but will typically be a liquid, gel, self-sustaining gel- matrix, or solid bait (e.g., tablets, granules, etc.). Exemplary carriers include, without limitation, agarose gel, gelatin gel, and/or pectin gel. In certain embodiments, the carrier is agarose gel, which is especially suited for aquatic habitats and breeding grounds. Insect baits are known in the art and are described, for example, in U.S. Pat. No. 8,841,272, which is incorporated herein by reference in its entirety.

[0162] The presently disclosed formulations can be present in the bait in an effective amount (i.e. , concentration) for the activity of the active agent, such as gene silencing. The concentration of the active agent in the bait may be about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% by weight of the bait. Any of these values may be used to define a range for the concentration of the active agent in the bait. For example, the concentration of the active agent in the bait may range from about 0.1 to about 1%, or from about 1 to about 5% by weight of the bait. The weight ratio of active agent to insect attractant (food) in the bait may be about 1: 1, 1 :5, 1 : 10, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, 1 : 100, 1 : 150 or 1 :200. Any of these values may be used to define a range for the weight ratio of the active agent to the insect attractant in the bait. For example, the weight ratio of the active agent to the insect attractant in the bait may be from about 1 :20 to about 1 :200, or from about 1 :50 to about 1: 100.

[0163] In certain embodiments, the concentration of a microparticle or nanoparticle comprising the at least one active agent and at least one modified PEI of the present disclosure in the bait may be about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% by weight of the bait. Any of these values may be used to define a range for the concentration of the microparticle or nanoparticle in the bait. For example, the concentration of the microparticle or nanoparticle in the bait may range from about 0.1 to about 1%, or from about 1 to about 5% by weight of the bait. The weight ratio of the microparticle or nanoparticle to insect attractant (food) in the bait may be about 1 : 1, 1 :5, 1 : 10, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, 1 : 100, 1 : 150 or 1 :200. Any of these values may be used to define a range for the weight ratio of the microparticle or nanoparticle to the insect attractant in the bait. For example, the weight ratio of the microparticle or nanoparticle to the insect attractant in the bait may be from about 1 :20 to about 1:200, or from about 1 :50 to about 1 : 100.

Herbicidal and Pesticidal Applications

[0164] In another aspect, the presently disclosed modified-based formulations can be used to deliver an active agent to target organisms for the purpose of killing and/or controlling the proliferation of the target organisms, such as insects, plant pathogens (e.g. , fungi, bacteria, viruses, and nematodes), and weeds. In certain embodiments, the presently disclosed aminolipidoid-based formulations comprise an insecticidal, nematidicidal, fungicidal, bacteriocidal, viricidal, or herbicidal active agent, or combinations thereof. In certain embodiments, these formulations are combined with an agriculturally acceptable carrier to form a insecticidal, nematodicidal, fungicidal, bacteriocidal, viricidal, or herbicidal formulation.

[0165] A target organism is an organism in which the presently disclosed herbicidal, insecticidal, or fungicidal formulations are intended to be functional, for example, to mediate gene silencing or suppression. In certain embodiments, a target organism is also a host organism, as described herein below. In other embodiments, a target organism is separate and distinct from a host organism that serves as a source of the active agent to be functional in the target organism.

[0166] The insecticidal, nematidicidal, fungicidal, bacteriocidal, viricidal, or herbicidal formulation may further be combined with an agriculturally acceptable carrier. The agriculturally acceptale carrier can be solid or liquid and is a substance useful in formulation of agricultural products, for example, fertilizers, herbicides, insecticides, fungicides, bactericides, viricides, and nematicides. Agriculturally acceptable carriers include, for example, natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, thickeners, binders or fertilizers. Such carriers are described for example, in WO 97/33890, which is incorporated herein by reference.

[0167] The presently disclosed formulations can be applied to the crop area or plant to be treated, simultaneously or in succession with further compounds. These further compounds can be, for example, fertilizers or micronutrient donors or other preparations, which influence the growth of plants. They can also be selective herbicides or no n- selective herbicides as well as insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, if desired together with further carriers, surfactants or application promoting adjuvants customarily employed in the art of formulation.

Screening of Capped Polymer Mixtures to Determine Ability to Transfect a Cell

[0168] In another aspect, the present disclosure provides for a method for screening capped polymer mixtures comprising: (1) reacting a polymer comprising at least one primary and/or secondary amino group with at least one cap precursor in at least two different ratios based on the total number of nitrogen atoms in the polymer to form at least two different mixtures of capped polymers; (2) separately combining each of the at least two different mixtures of capped polymers with an oligo- or polynucleotide to form at least two different mixtures of oligo- or polynucleotide/capped polymer complexes; and (3) separately assaying each of the at least two different mixtures of oligo- or polynucleotide/capped polymer complexes to determine the ability of each mixture to transfect a cell with the oligo- or polynucleotide. As used herein, the phrase "capped polymer" means any polymer comprising at least one primary and/or secondary amino group which has been reacted with at least one cap precursor. As used herein, the phrase "cap precursor" means any molecule that contains at least one functional group capable of reacting with a primary and/or secondary amino group of a polymer. When the primary and/or secondary amino group of the polymer is reacted with the cap precursor, the resulting moiety derived from the molecule is a "cap" and the primary and/or secondary amino group is deemed "capped."

[0169] In certain embodiments, the above method for screening further comprises: (4) selecting an oligo- or polynucleotide/capped polymer complex mixture determined to be able to transfect a cell with the oligo- or polynucleotide; (5) separating the mixture into at least two oligo- or polynucleotide/capped polymer complex sub-mixtures on the basis of average molecular weight; and (6) separately assaying each of the at least two different sub-mixtures of to determine the ability of each sub-mixture to transfect a cell with the oligo- or polynucleotide.

[0170] The polymer and the at least one cap precursor can be reacted in any number of suitable ratios at least two different ratios to form different mixtures of capped polymers. Examples of such numbers of suitable ratios include, but are not limited to, 2 different ratios, 3 different ratios, 4 different ratios, 5 different ratios, 6 different ratios, 7 different ratios, 8 different ratios, 9 different ratios, and 10 different ratios. In certain embodiments, the polymer and the at least one cap precursor are reacted in three different ratios to form three different mixtures of capped polymers. In each instance, the polymer and the at least one cap precursor can be reacted in any suitable ratios. Examples of such suitable ratios include, but are not limited to, 1: 1, 1;2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10, 2;1, 2:3, 2:5, 2:7, 2:9, 3: 1, 3:2, 3:4, 3:5, 3:7, 3:8, 3: 10, 4: 1, 4:3, 4:5, 4:7, 5: 1, 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 6: 1, 6:5, 6:7, 7: 1, 7:2, 7:3, 7:4, 7:5, 7:6, 7:8, 7;9, 7: 10, 8: 1, 8:3, 8:5, 8:7, 8:9, 9: 1, 9:2, 9:4, 9:5, 9:7, 9: 10, 10: 1, 10:3, 10;7, and 10:9. In certain embodiments the three different polymer/cap precursor ratios are 3:3, 3:2, and 3: 1.

[0171] In certain embodiments of the above method for screening the polymer comprising at least one primary and/or secondary amino group is a polyethyleneimine prepared by the ring-opening polymerization of aziridine, a polyethyleneimine derived from the partial or complete hydrolysis of a poly(2-alkyl-2-oxazoline), or combinations thereof. In a certain embodiment, the polyethyleneimine is derived from the partial or complete hydrolysis of poly(2-ethyl-2-oxazoline). [0172] In certain embodiments, the at least one cap precursor is a compound of (Ilia):

wherein

R₅ is hydrogen, an optionally substituted aliphatic or

cycloaliphatic group, an optionally substituted hetero aliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group; and

R₆ is an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted hetero aliphatic or

heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group; a compound of formula (IVa):

wherein

are, independently, hydrogen or an optionally substituted aliphatic group;

Rg is an optionally substituted aliphatic group; and

X is O or N; or any combination thereof.

[0173] In some embodiments , the oligo- or polynucleotide used in the above method for screening reduces the expression of a target gene expressed by the cell. As used herein, the term "expression" is defined as the process by which a polypeptide is produced from DNA. This process involves the transcription of the gene into mRNA and the translation of this mRNA into a polypeptide. Thus, "expression" may refer to the production of RNA, or protein, or both. As used herein, term "target gene" is defined as a gene in which the expression is regulated by the oligo- or polynucleotide.

[0174] The assaying step in the above method for screening can comprise measuring the expression level of the target gene. As used herein, the term "expression level" is defined as the level of mRNA, as well as pre-mRNA nascent transcript(s), transcript processing intermediates, mature mRNA(s) and degradation products, or the level of a protein, encoded by the gene in the cell. In certain embodiments, the assaying step comprises measuring mRNA levels of the target gene. The mRNA levels may be measured by any method known in the art. Examples of such methods include, but are not limited to, Northern blot analyses, polymerase chain reaction analyses, and probe arrays. One diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

Hybridization of an mRNA with the probe indicates that the target gene in question is being expressed.

[0175] In certain embodiments, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In certain other embodiments, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by a target gene of the present invention.

[0176] In certain embodiments, the method for determining the level of mRNA in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88: 189-193), self sustained sequence replication (Guatelli et al, 1990, Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh et al, 1989, Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6: 1197), rolling circle replication (Lizardi et al., U.S. Patent No. 5,854,033), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice- versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. In certain embodiments, the level of mRNA is determined by quantitative PCR, for example, by real-time quantitative PCR.

[0177] For in situ methods, mRNA does not need to be isolated from the cell prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the target gene.

[0178] As an alternative to making determinations based on the absolute expression level of the target gene, determinations may be based on the normalized expression level of the target gene. Expression levels are normalized by correcting the absolute expression level of a target gene by comparing its expression to the expression of a gene that is not the target gene, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., one mixture of capped polymers and the oligo- or polynucleotide, to another sample, e.g., a different mixture of the capped polymers and the oligo- or polynucleotide.

[0179] In certain embodiments of the above method for screening, the assaying comprises measuring expression and/or activity of a protein encoded by the target gene. As used herein, the term "activity of a protein" is defined as the amount of protein activity, typically enzymatic activity, as determined by a quantitative, semi-quantitative, or qualitative assay. Activity is typically determined by monitoring the amount of product produced in an assay using a substrate that produces a readily detectable product, e.g., colored product, fluorescent product, or radioactive product. The specific assay performed depends, for example, on the activity to be measured.

[0180] One agent for detecting protein expression is an antibody capable of binding to such a protein or a fragment thereof, preferably an antibody with a detectable label.

Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment or derivative thereof (e.g., Fab or F(ab')₂) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

[0181] Proteins from cells can be isolated using techniques that are well known to those of skill in the art. The protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

[0182] A variety of formats can be employed to determine the level of a protein that binds to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), western blot analysis and enzyme linked immunoabsorbant assay (ELISA). A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cells express the target gene.

[0183] In one format, antibodies, or antibody fragments or derivatives, can be used in methods such as western blots or immunofluorescence techniques to detect the expressed proteins. In such uses, it is generally preferable to immobilize either the antibody or proteins on a solid support. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. [0184] One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present invention. For example, protein isolated from the cells can be run on a polyacrylamide gel

electrophoresis and immobilized onto a solid phase support such as nitrocellulose. The support can then be washed with suitable buffers followed by treatment with the detectably labeled antibody. The solid phase support can then be washed with the buffer a second time to remove unbound detectably labeled antibody. The amount of bound labeled antibody on the solid support can then be detected by conventional means.

[0185] In a particular embodiment, the expression of the protein is measured by a method selected from Western blot analysis, flow cytometry, ELISA, and mass spectrometry.

[0186] The target gene in the above methods for screening may be any gene that is expressed by the cell. In certain embodiments, the target gene is endogenous to the cell. In other embodiments, the target gene is heterologous to the cell. For example, the cell may be transfected with a target gene encoding a marker protein. In certain embodiments, the marker protein is a fluorescent marker protein. Suitable marker proteins include, but are not limited to, Renilla luciferase, Firefly luciferase, and green fluorescent protein (GFP).

[0187] Any type of cell that is capable of being transfected may be used in the above methods for screening, including but not limited to a plant cell, an insect cell, a bacterial cell, a fungal cell and a mammalian cell.

[0188] Reduced expression of the target gene, or reduced activity of a protein encoded by the target gene, in response to treatment with a mixture of the oligo- or polynucleotide and the capped polymer complex indicates that the mixture is able to transfect the cell. In certain embodiments, the mixture reduces expression of the target gene by at least about 50%, 60%, 70%, 80%, 90% or 95% relative to expression of a the target gene in a cell that is not treated with the mixture. In certain embodiments, the mixture reduces activity of a protein encoded by the target gene by at least about 50%, 60%, 70%, 80%, 90% or 95% relative to the activity of the protein in a cell that is not treated with the mixture.

[0189] For example, in a particular embodiment, the cell is transfected with a target gene encoding Firefly luciferase and an additional gene encoding Renilla luciferase. The transfected cells are treated with at least two different mixtures comprising a capped polymer complex and an oligo- or polynucleotide (e.g. , an siRNA) that reduces expression of the Firefly luciferase but does not significantly alter expression of the Renilla luciferase.

Luciferase and Renilla gene expression may be measured by a luminescence assay (e.g. , Dual Glow Assay, Pro mega, Madison, Wis.). Mixtures of a capped polymer complex and an oligo- or polynucleotide that reduce expression of the Firefly luciferase but do not significantly alter expression of the Renilla luciferase are determined to be able to transfect a cell.

[0190] The ability of the mixture of the capped polymer complex and the oligo- or polynucleotide to transfect a cell may also be determined by determining a phenotype of the cell, or a phenotype of an organism comprising the cell, after treatment of the cell or organism with the mixture. Examples of phenotypes that may be determined include, but are not limited to, changes in growth, viability, herbicide tolerance, insect tolerance, disease tolerance.

[0191] Any oligo- or polynucleotide that reduces expression of the target gene may be used in the aforementioned methods, including but not limited to interfering RNA molecules (e.g., siRNA, aiRNA, miRNA), and antisense RNA. In a particular embodiment, the oligo- or polynucleotide is an siRNA or an antisense RNA.

Active Agents

[0192] Active agents that can be delivered to a target organism using the presently disclosed formulations include, but are not limited to, any type of molecule or compound including, but not limited to, nucleic acids, peptides, polypeptides, small molecules, and mixtures thereof. Examples of nucleic acids include, but are not limited to, interfering RNA molecules (e.g. , siRNA, aiRNA, miRNA), antisense oligonucleotides, plasmids, ribozymes, immuno stimulatory oligonucleotides, and mixtures thereof.

[0193] In certain embodiments, the active agent comprises a nucleic acid. In certain embodiments, the nucleic acid comprises an interfering RNA molecule such as, e.g., an siRNA, aiRNA, miRNA, or mixtures thereof. In certain embodiments, the nucleic acid comprises single- stranded or double- stranded DNA, RNA, or a DNA/RNA hybrid such as, e.g. , an antisense oligonucleotide, a ribozyme, a plasmid, an immuno stimulatory

oligonucleotide, or mixtures thereof.

I l l [0194] In certain embodiments, presently disclosed particles are associated with a nucleic acid. In some embodiments, the nucleic acid is fully encapsulated in a lipidoid particle. As used herein, the term "nucleic acid" includes any oligonucleotide or

polynucleotide, with fragments containing up to 60 nucleotides generally termed

oligonucleotides, and longer fragments termed polynucleotides. In particular embodiments, oligonucleotides of the invention are about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 nucleotides in length. Any of these values may be used to define a range for the size of the oligonucleotide. For example, the size of the oligonucleotide may range from 15-60, 20-60 or 25-60 nucleotides in length. In particular embodiments, the polynucleotide is 65, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more nucleotides in length. In particular embodiments, the polynucleotide is at least 65, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 nucleotides in length. Any of these values may be used to define a range for the size of the polynucleotide. For example, the polynucleotide may range from 100- 150, 150-200, 200-250, 250-300, 300-350, 350-400, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900- 950, or 950- 1000 nucleotides in length. The nucleic acid may be administered alone in the particles of the present disclosure, or in combination (e.g. , co-administered) with particles of the present disclosure comprising peptides, polypeptides, or small molecules, such as conventional drugs.

[0195] In the context of this invention, the terms "polynucleotide" and

"oligonucleotide" refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally-occurring bases, sugars, and intersugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.

[0196] Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleo tides. A deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5' and 3' carbons of this sugar to form an alternating, unbranched polymer. A ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose.

[0197] Nucleic acids that can be used in the presently disclosed formulations includes any form of nucleic acid that is known. The nucleic acids used herein can be single- stranded DNA or RNA, or double- stranded DNA or RNA, or DNA-RNA hybrids. Examples of double- stranded DNA are described herein and include, e.g. , structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double- stranded RNA are described herein and include, e.g. , siRNA and other RNAi agents such as aiRNA and pre-miRNA. Single-stranded nucleic acids include, e.g., antisense oligonucleotides, ribozymes, mature miRNA, and triplex-forming oligonucleotides.

[0198] Nucleic acids that can be used in the formulations of the present disclosure may be of various lengths, which is generally dependent upon the particular form of nucleic acid. For example, in certain embodiments, plasmids or genes may be from about 1,000 to about 100,000 nucleotide residues in length. In certain embodiments, oligonucleotides may range from about 10 to about 100 nucleotides in length. In certain embodiments,

oligonucleotides, both single- stranded, double- stranded, and triple- stranded, may range in length from about 10 to about 60 nucleotides, from about 15 to about 60 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, or from about 20 to about 30 nucleotides in length.

[0199] In certain embodiments, an oligonucleotide (or a strand thereof) that can be used in the presently disclosed formulations specifically hybridizes to or is complementary to a target polynucleotide sequence. The terms "specifically hybridizable" and

"complementary" as used herein indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. In certain embodiments, an oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target sequence interferes with the normal function of the target sequence to cause a loss of utility or expression therefrom, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired. Thus, the oligonucleotide may include 1, 2, 3, or more base substitutions as compared to the region of a gene or mRNA sequence that it is targeting or to which it specifically hybridizes.

[0200] In certain embodiments, the oligo- or polynucleotide is optionally purified and substantially pure. In some embodiments, the polynucleotide is greater than 50% pure. In some embodiments, the oligo- or polynucleotide is greater than 75% pure. In some embodiments, the oligo- or polynucleotide is greater than 95% pure. The oligo- or polynucleotide may be provided by any means known in the art. In certain embodiments, the oligo- or polynucleotide has been engineered using recombinant techniques. The oligo- or polynucleotide may also be obtained from natural sources and purified from contaminating components found normally in nature. The oligo- or polynucleotide may also be chemically synthesized in a laboratory. In certain embodiments, the oligo- or polynucleotide is synthesized using standard solid phase chemistry.

[0201] The oligo- or polynucleotide may be modified by chemical or biological means. In certain embodiments, these modifications lead to increased stability of the oligo- or polynucleotide. Examples of such modifications include, but are not limited to, methylation, phosphorylation, and end-capping.

[0202] The oligo- or polynucleotide to be delivered may be in any form. Examples of such forms include, but are not limited to, a circular plasmid, a linearized plasmid, a cosmid, a viral genome, a modified viral genome, an artificial chromosome, dsRNA, ssRNA, dsDNA, ssDNA, RNA/DNA hybrids, dsRNA hairpins, siRNA, aiRNA, and miRNA.

[0203] The oligo- or polynucleotide may be of any sequence. In certain

embodiments, the oligo- or polynucleotide encodes a protein or peptide. The encoded proteins may be enzymes, structural proteins, receptors, soluble receptors, ion channels, or cytokines. The oligo- or polynucleotide may also comprise regulatory regions to control the expression of a gene. These regulatory regions may include, but are not limited to, promoters, enhancer elements, repressor elements, TATA box, ribosomal binding sites, and stop site for transcription. In certain embodiments, the polynucleotide is not intended to encode a protein. For example, the polynucleotide may be used to fix an error in the genome of the cell being transfected.

[0204] In certain embodiments, the nucleic acid is modified. As used herein, the term

"modified" in reference to a nucleic acid (e.g. , an oligonucleotide or polynucleotide) is defined as a nucleic acid that contains variations of the standard bases, sugars and/or phosphate backbone chemical structures occurring in ribonucleic (i.e. , A, C, G and U) and deoxyribonucleic (i.e. , A, C, G and T) acids. Particular modifications of nucleic acids are further described below.

[0205] In certain embodiments, the oligo- or polynucleotide is an RNA that carries out RNA interference (RNAi). The term "interfering RNA" or "RNAi" or "interfering RNA sequence" refers to single- stranded RNA (e.g. , mature miRNA) or double- stranded RNA (e.g., duplex RNA, such as siRNA, aiRNA, or pre-miRNA) that is capable of reducing or inhibiting the expression of a target gene or sequence (e.g., by mediating the degradation or inhibiting the translation of mRNAs which are complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence.

Interfering RNA thus refers to the single- stranded RNA that is complementary to a target mRNA sequence or to the double- stranded RNA formed by two complementary strands or by a single, self-complementary strand. Interfering RNA may have substantial or complete identity to the target gene or sequence, or may comprise a region of mismatch (i.e. , a mismatch motif). The sequence of the interfering RNA can correspond to the full-length target gene, or a subsequence thereof. siRNA

[0206] In certain embodiments, the active agent comprises an siRNA. The siRNA molecule can comprise a double- stranded region of about 15 to about 60 nucleotides in length (e.g., about 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 25, or 19 to 25 nucleotides in length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). The siRNA molecules used in the presently disclosed formulations are capable of silencing the expression of a target sequence in vitro and/or in vivo.

[0207] In certain embodiments, the siRNA molecule comprises modified nucleotides including, but not limited to, 2'-0-methyl (2'OMe) nucleotides, 2'-deoxy-2'-fluoro(2'F) nucleotides, 2'-deoxy nucleotides, 2'-0-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof. In other embodiments, the siRNA comprises 2'OMe nucleotides (e.g. , 2'OMe purine and/or pyrimidine nucleotides) such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, 2'OMe-cytosine nucleotides, and mixtures thereof. In certain embodiments, the siRNA does not comprise 2'OMe-cytosine nucleotides. In certain embodiments, the siRNA comprises a hairpin loop structure.

[0208] In certain embodiments, the siRNA may comprise modified nucleotides in one strand (i.e. , sense or antisense) or both strands of the double- stranded region of the siRNA molecule. In certain embodiments, uridine and/or guanosine nucleotides are modified at selective positions in the double- stranded region of the siRNA duplex. With regard to uridine nucleotide modifications, at least one, two, three, four, five, six, or more of the uridine nucleotides in the sense and/or antisense strand can be a modified uridine nucleotide such as a 2'OMe-uridine nucleotide. In certain embodiments, every uridine nucleotide in the sense and/or antisense strand is a 2'OMe-uridine nucleotide. With regard to guanosine nucleotide modifications, at least one, two, three, four, five, six, or more of the guanosine nucleotides in the sense and/or antisense strand can be a modified guanosine nucleotide such as a 2'OMe- guanosine nucleotide. In certain embodiments, every guanosine nucleotide in the sense and/or antisense strand is a 2'OMe-guanosine nucleotide.

[0209] In certain embodiments, at least one, two, three, four, five, six, seven, or more

5'-GU-3' motifs in an siRNA sequence may be modified, e.g., by introducing mismatches to eliminate the 5'-GU-3' motifs and/or by introducing modified nucleotides such as 2'OMe nucleotides. The 5'-GU-3' motif can be in the sense strand, the antisense strand, or both strands of the siRNA sequence. The 5'-GU-3 ' motifs may be adjacent to each other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides.

[0210] In certain embodiments, a modified siRNA molecule is capable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the expression of the target sequence relative to the corresponding unmodified siRNA sequence.

[0211] In certain embodiments, the siRNA molecule does not comprise phosphate backbone modifications, e.g. , in the sense and/or antisense strand of the double- stranded region. In certain embodiments, the siRNA comprises one, two, three, four, or more phosphate backbone modifications, e.g. , in the sense and/or antisense strand of the double- stranded region. In certain embodiments, the siRNA does not comprise phosphate backbone modifications. [0212] In certain embodiments, the siRNA does not comprise 2'-deoxy nucleotides, e.g. , in the sense and/or antisense strand of the double- stranded region. In certain

embodiments, the siRNA comprises one, two, three, four, or more 2'-deoxy nucleotides, e.g., in the sense and/or antisense strand of the double- stranded region. In certain embodiments, the siRNA does not comprise 2'-deoxy nucleotides.

[0213] In certain embodiments, the nucleotide at the 3 '-end of the double- stranded region in the sense and/or antisense strand is not a modified nucleotide. In certain embodiments, the nucleotides near the 3 '-end (e.g. , within one, two, three, or four nucleotides of the 3 '-end) of the double- stranded region in the sense and/or antisense strand are not modified nucleotides.

[0214] The siRNA molecules described herein may have 3' overhangs of one, two, three, four, or more nucleotides on one or both sides of the double- stranded region, or may lack overhangs (i.e. , have blunt ends) on one or both sides of the double- stranded region. In certain embodiments, the siRNA has 3 ' overhangs of two nucleotides on each side of the double- stranded region. In certain embodiments, the 3 ' overhang on the antisense strand has complementarity to the target sequence and the 3' overhang on the sense strand has complementarity to a complementary strand of the target sequence. Alternatively, the 3' overhangs do not have complementarity to the target sequence or the complementary strand thereof. In certain embodiments, the 3' overhangs comprise one, two, three, four, or more nucleotides such as 2'-deoxy(2'H) nucleotides. In certain embodiments, the 3' overhangs comprise deoxythymidine (dT) and/or uridine nucleotides. In certain embodiments, one or more of the nucleotides in the 3' overhangs on one or both sides of the double- stranded region comprise modified nucleotides. Examples of modified nucleotides are described above and include, but are not limited to, 2'OMe nucleotides, 2'-deoxy-2'F nucleotides, 2'-deoxy nucleotides, 2'-0-2-MOE nucleotides, LNA nucleotides, and mixtures thereof. In certain embodiments, one, two, three, four, or more nucleotides in the 3 ' overhangs present on the sense and/or antisense strand of the siRNA comprise 2'OMe nucleotides (e.g. , 2'OMe purine and/or pyrimidine nucleotides) such as, for example, 2'OMe-guanosine nucleotides, 2'OMe- uridine nucleotides, 2'OMe-adenosine nucleotides, 2'OMe-cytosine nucleotides, and mixtures thereof.

[0215] The siRNA may comprise at least one or a cocktail (e.g. , at least two, three, four, five, six, seven, eight, nine, ten, or more) of unmodified and/or modified siRNA sequences that silence target gene expression. The cocktail of siRNA may comprise sequences, which are directed to the same region or domain (e.g., a "hot spot") and/or to different regions or domains of one or more target genes. In certain embodiments, one or more (e.g. , at least two, three, four, five, six, seven, eight, nine, ten, or more) modified siRNA that silence target gene expression are present in a cocktail. In certain embodiments, one or more (e.g. , at least two, three, four, five, six, seven, eight, nine, ten, or more) unmodified siRNA sequences that silence target gene expression are present in a cocktail.

[0216] In certain embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence or a portion thereof. In certain embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that is 100% complementary to the target sequence or a portion thereof. In certain embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that specifically hybridizes to the target sequence or a portion thereof.

[0217] In certain embodiments, the sense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the target sequence or a portion thereof. In certain embodiments, the sense strand of the siRNA molecule comprises or consists of a sequence that is 100% identical to the target sequence or a portion thereof.

[0218] The siRNA that can be used in the presently disclosed formulations are capable of silencing the expression of a target gene of interest. Each strand of the siRNA duplex can be about 15 to about 60 nucleotides in length, or about 15 to about 30 nucleotides in length. In certain embodiments, the siRNA comprises at least one modified nucleotide. In some embodiments, the modified siRNA contains at least one 2'OMe purine or pyrimidine nucleotide such as a 2'OMe-guanosine, 2'OMe-uridine, 2'OMe-adenosine, and/or 2'OMe- cytosine nucleotide. In certain embodiments, one or more of the uridine and/or guanosine nucleotides are modified. The modified nucleotides can be present in one strand (i.e., sense or antisense) or both strands of the siRNA. The siRNA sequences may have overhangs or may lack overhangs (i.e. , have blunt ends).

[0219] The modified siRNA generally comprises from about 1% to about 100% (e.g. , about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 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%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the double- stranded region of the siRNA duplex. In certain embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or more of the nucleotides in the double- stranded region of the siRNA comprise modified nucleotides.

[0220] In certain embodiments, less than about 25% (e.g., less than about 25%, 24%,

23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of the nucleotides in the double- stranded region of the siRNA comprise modified nucleotides.

[0221] In certain embodiments, from about 1% to about 25% (e.g., from about 1%-

25%, 2%-25%, 3%-25%, 4%-25%, 5%-25%, 6%-25%, 7%-25%, 8%-25%, 9%-25%, 10%- 25%, ll%-25%, 12%-25%, 13%-25%, 14%-25%, 15%-25%, 16%-25%, 17%-25%, 18%- 25%, 19%-25%, 20%-25%, 21%-25%, 22%-25%, 23%-25%, 24%-25%, etc.) or from about 1% to about 20% (e.g., from about l%-20%, 2%-20%, 3%-20%, 4%-20%, 5%-20%, 6%- 20%, 7%-20%, 8%-20%, 9%-20%, 10%-20%, ll%-20%, 12%-20%, 13%-20%, 14%-20%, 15%-20%, 16%-20%, 17%-20%, 18%-20%, 19%-20%, 1%-19%, 2%-19%, 3%-19%, 4%- 19%, 5%-19%, 6%-19%, 7%-19%, 8%-19%, 9%-19%, 10%-19%, 11%-19%, 12%-19%, 13%-19%, 14%-19%, 15%-19%, 16%-19%, 17%-19%, 18%-19%, 1%-18%, 2%-18%, 3%- 18%, 4%-18%, 5%-18%, 6%-18%, 7%-18%, 8%-18%, 9%-18%, 10%-18%, 11%-18%, 12%- 18%, 13%-18%, 14%-18%, 15%-18%, 16%-18%, 17%-18%, 1%-17%, 2%-17%, 3%-17%, 4%-17%, 5%-17%, 6%-17%, 7%-17%, 8%-17%, 9%-17%, 10%- 17%, 11%- 17%, 12%- 17%, 13%- 17%, 14%- 17%, 15%- 17%, 16%- 17%, 1%-16%, 2%-16%, 3%-16%, 4%-16%, 5%- 16%, 6%-16%, 7%-16%, 8%-16%, 9%-16%, 10%-16%, 11%-16%, 12%-16%, 13%-16%, 14%- 16%, 15%- 16%, 1%-15%, 2%-15%, 3%-15%, 4%-15%, 5%-15%, 6%-15%, 7%-15%, 8%-15%, 9%-15%, 10%-15%, 11%-15%, 12%-15%, 13%-15%, 14%- 15%, etc.) of the nucleotides in the double- stranded region of the siRNA comprise modified nucleotides.

[0222] In certain embodiments, e.g., when one or both strands of the siRNA are selectively modified at uridine and/or guanosine nucleotides, the resulting modified siRNA can comprise less than about 30% modified nucleotides (e.g., less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% modified nucleotides) or from about 1% to about 30% modified nucleotides (e.g., from about l%-30%, 2%-30%, 3%-30%, 4%-30%, 5%-30%, 6%-30%, 7%-30%, 8%-30%, 9%-30%, 10%-30%, l l%-30%, 12%-30%, 13%-30%, 14%-30%, 15%-30%, 16%-30%, 17%-30%, 18%-30%, 19%-30%, 20%-30%, 21%-30%, 22%-30%, 23%-30%, 24%-30%, 25%-30%, 26%-30%, 27%-30%, 28%-30%, or 29%-30% modified nucleotides).

[0223] Examples of modified nucleotides suitable for use in the presently disclosed formulations include, but are not limited to, ribonucleotides having a 2'-0-methyl (2'OMe), 2'-deoxy-2'-fluoro(2'F), 2'-deoxy, 5-C-methyl, 2'-0-(2-methoxyethyl) (MOE), 4'-thio, 2'- amino, or 2'-C-allyl group. Modified nucleotides having a Northern conformation are also suitable for use in siRNA molecules. Such modified nucleotides include, without limitation, locked nucleic acid (LNA) nucleotides (e.g., 2'-0, 4'-C-methylene-(D-ribofuranosyl) nucleotides), 2'-0-(2-methoxyethyl) (MOE) nucleotides, 2'-methyl-thio-ethyl nucleotides, 2'- deoxy-2'-fluoro(2'F) nucleotides, 2'-deoxy-2'-chloro(2'Cl) nucleotides, and 2'-azido nucleotides. In certain instances, the siRNA molecules described herein include one or more G-clamp nucleotides. A G-clamp nucleotide refers to a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine nucleotide within a duplex. In addition, nucleotides having a nucleotide base analog such as, for example, C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4- nitroindole, 5-nitroindole, and 6-nitroindole can be incorporated into siRNA molecules.

[0224] In certain embodiments, the siRNA molecules may further comprise one or more chemical modifications such as terminal cap moieties, phosphate backbone

modifications, and the like. Examples of terminal cap moieties include, but are not limited to, inverted deoxy abasic residues, glyceryl modifications, 4',5'-methylene nucleotides, 1-(β- D-erythrofuranosyl) nucleotides, 4'-thio nucleotides, carbocyclic nucleotides, 1,5- anhydrohexitol nucleotides, L-nucleotides, a-nucleotides, modified base nucleotides, threo- pentofuranosyl nucleotides, acyclic 3 ',4'-seco nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3 '-3 '-inverted nucleotide moieties, 3'- 3 '-inverted abasic moieties, 3'-2'-inverted nucleotide moieties, 3'-2'-inverted abasic moieties, 5 '-5 '-inverted nucleotide moieties, 5 '-5 '-inverted abasic moieties, 3 '-5 '-inverted deoxy abasic moieties, 5 '-amino- alky 1 phosphate, l,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydro xypropyl phosphate, 1,4-butanediol phosphate, 3'-phosphoramidate, 5'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 5'-amino, 3'-phosphorothioate, 5'-phosphorothioate, phosphorodithioate, and bridging or non-bridging methylphosphonate or 5'-mercapto moieties. Examples of phosphate backbone modifications (i.e., resulting in modified internucleotide linkages) include, but are not limited to, phosphorothioate,

phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions. Such chemical modifications can occur at the 5'- end and/or 3 '-end of the sense strand, antisense strand, or both strands of the siRNA.

[0225] In certain embodiments, the sense and/or antisense strand of the siRNA molecule can further comprise a 3'-terminal overhang having about 1 to about 4 (e.g., 1, 2, 3, or 4) 2'-deoxy ribonucleotides and/or any combination of modified and unmodified nucleotides.

[0226] The siRNA molecules can optionally comprise one or more non-nucleotides in one or both strands of the siRNA. As used herein, the term "non-nucleotide" refers to any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base such as adenosine, guanine, cytosine, uracil, or thymine and therefore lacks a base at the 1 '-position.

[0227] In certain embodiments, chemical modification of the siRNA comprises attaching a conjugate to the siRNA molecule. The conjugate can be attached at the 5' and/or 3 '-end of the sense and/or antisense strand of the siRNA via a covalent attachment such as, e.g., a biodegradable linker. The conjugate can also be attached to the siRNA, e.g., through a carbamate group or other linking group. In certain instances, the conjugate is a molecule that facilitates the delivery of the siRNA into a cell. aiRNA

[0228] In certain embodiments, the active agent comprises an asymmetrical interfering RNA (aiRNA). In certain embodiments, aiRNA duplexes of various lengths may be designed with overhangs at the 3' and 5' ends of the antisense strand to target an mRNA of interest. In certain embodiments, the sense strand of the aiRNA molecule is about 10-25, 12- 20, 12- 19, 12- 18, 13- 17, or 14- 17 nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain embodiments, the antisense strand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotides in length, or about 15-30, 15-25, or 19-25 nucleotides in length, or about 20-24, 21-22, or 21-23 nucleotides in length.

[0229] In certain embodiments, the 5' antisense overhang contains one, two, three, four, or more nontargeting nucleotides (e.g. , "AA", "UU", "dTdT", etc.). In other

embodiments, the 3' antisense overhang contains one, two, three, four, or more nontargeting nucleotides (e.g., "AA", "UU", "dTdT", etc.). In certain embodiments, the aiRNA molecules described herein may comprise one or more modified nucleotides, e.g. , in the double- stranded (duplex) region and/or in the antisense overhangs. As a non-limiting example, aiRNA sequences may comprise one or more of the modified nucleotides described above for siRNA sequences. In certain embodiments, the aiRNA molecule comprises 2'OMe nucleotides such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, or mixtures thereof.

[0230] In certain embodiments, aiRNA molecules may comprise an antisense strand which corresponds to the antisense strand of an siRNA molecule, e.g. , one of the siRNA molecules described herein. In certain embodiments, aiRNA molecules may be used to silence the expression of any of a target gene.

[0231] In certain embodiments, the aiRNA molecule comprises a double- stranded

(duplex) region of about 10 to about 25 (base paired) nucleotides in length, wherein the aiRNA molecule comprises an antisense strand comprising 5' and 3' overhangs, and wherein the aiRNA molecule is capable of silencing target gene expression.

[0232] In certain embodiments, each of the 5' and 3 ' overhangs on the antisense strand comprises or consists of one, two, three, four, five, six, seven, or more nucleotides.

[0233] In certain embodiments, the aiRNA molecule comprises modified nucleotides selected from the group consisting of 2'OMe nucleotides, 2'F nucleotides, 2'-deoxy nucleotides, 2'-0-MOE nucleotides, LNA nucleotides, and mixtures thereof. miRNA

[0234] In certain embodiments, the active agent comprises a micro RNAs (miRNA).

Generally, miRNA are single- stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression. In certain embodiments, the miRNA molecules described herein are about 15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotides in length, or about 15-30, 15-25, or 19-25 nucleotides in length, or about 20-24, 21-22, or 21-23 nucleotides in length. In certain embodiments, the miRNA molecule comprises about 15 to about 60 nucleotides in length, wherein the miRNA molecule is capable of silencing target gene expression.

[0235] In certain embodiments, miRNA molecules may comprise one or more modified nucleotides. As a non-limiting example, miRNA sequences may comprise one or more of the modified nucleotides described above for siRNA sequences. In certain embodiments, the miRNA molecule comprises 2'OMe nucleotides such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, or mixtures thereof. In certain embodiments, the miRNA molecule comprises modified nucleotides selected from the group consisting of 2'F nucleotides, 2'-deoxy nucleotides, 2'-0-MOE nucleotides, LNA nucleotides, and mixtures thereof. dsRNA

[0236] In certain embodiments, the active agent is a dsRNA (double- stranded RNA).

In certain embodiments, the active agent is an shRNA (short hairpin RNA).

Antisense Polynucleotide

[0237] In certain embodiments, the active agent is an antisense oligonucleotide. The terms "antisense polynucleotide" or "antisense" include polynucleotides that are

complementary to a targeted polynucleotide sequence. Antisense polynucleotides are single strands of DNA or RNA that are complementary to a chosen sequence.

[0238] In certain embodiments, the polynucleotide is an antisense RNA. Antisense

RNA polynucleotides prevent the translation of complementary RNA strands by binding to the RNA. Antisense DNA polynucleotides can be used to target a specific, complementary (coding or non-coding) RNA. If binding occurs, this DNA/RNA hybrid can be degraded by the enzyme RNase H. In certain embodiments, antisense polynucleotides comprise from about 10 to about 60 nucleotides, or from about 15 to about 30 nucleotides. The term also encompasses antisense polynucleotides that may not be exactly complementary to the desired target gene. Thus, the invention can be utilized in instances where non-target specific - activities are found with antisense, or where an antisense sequence containing one or more mismatches with the target sequence is the most preferred for a particular use.

[0239] Methods of producing antisense polynucleotides are known in the art and can be readily adapted to produce an antisense polynucleotides that targets any polynucleotide sequence. Selection of antisense polynucleotide sequences specific for a given target sequence is based upon analysis of the chosen target sequence and determination of secondary structure, Tm, binding energy, and relative stability. Antisense polynucleotides may be selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell. Highly preferred target regions of the mRNA include those regions at or near the AUG translation initiation codon and those sequences that are substantially complementary to 5' regions of the mRNA. These secondary structure analyses and target site selection considerations can be performed, for example, using v.4 of the OLIGO primer analysis software (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithm software

(Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

Ribozymes

[0240] In certain embodiments, the active agent is a ribozyme. Ribozymes are RNA- protein complexes having specific catalytic domains that possess endonuclease activity. For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate. This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

[0241] The enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, hepatitis δ virus, group I intron or RNaseP RNA (in association with an RNA guide sequence), or Neurospora VS RNA motif, for example. Important characteristics of enzymatic nucleic acid molecules used according to the invention are that they have a specific substrate binding site which is complementary to one or more of the target gene DNA or RNA regions, and that they have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule. [0242] Methods of producing a ribozyme targeted to any polynucleotide sequence are known in the art. Ribozyme activity can be optimized by altering the length of the ribozyme binding arms or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases, modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.

Insecticides

[0243] In certain embodiments, an insecticide for killing or controlling the proliferation of an insect is combined with one of the active agents described above.

Examples of suitable insecticides include, but are not limited to, those provided in Table 1.

[0244] Table 1.

[0245] Additional non-limiting examples of suitable insecticides include biologies, hormones or pheromones such as azadirachtin, Bacillus species, Beauveria species, codlemone, Metarrhizium species, Paecilomyces species, thuringiensis and Verticillium species, and active compounds having unknown or non-specified mechanisms of action such as fumigants (such as aluminium phosphide, methyl bromide and sulphuryl fluoride) and selective feeding inhibitors (such as cryolite, flonicamid and pymetrozine). Examples of mite growth inhibitors include, but are not limited to, clofentezine, etoxazole and hexythiazox, amidoflumet, benclothiaz, benzoximate, bifenazate, bromopropylate, buprofezin,

chinomethioat, chlordimeform, chlorobenzilate, chloropicrin, clothiazoben, cycloprene, cyflumetofen, dicyclanil, fenoxacrim, fentrifanil, flubenzimine, flufenerim, flutenzin, gossyplure, hydramethylnone, japonilure, metoxadiazone, petroleum, piperonyl butoxide, potassium oleate, pyrafluprole, pyridalyl, pyriprole, sulfluramid, tetradifon, tetrasul, triarathene, verbutin, furthermore the compound 3-methylphenyl propylcarbamate

(Tsumacide Z), the compound 3-(5-chloro-3-pyridinyl)-8-(2,2,2-trifluoroethyl)-8- azabicyclo[3.2.1]octa- ne-3-carbonitrile (CAS reg. No. 185982-80-3) and the corresponding 3-endo isomer (CAS reg. No. 185984-60-5) (cf. WO 96/37494, WO 98/25923), and also preparations comprising insecticidally effective plant extracts, nematodes, fungi, or viruses.

Herbicides

[0246] In certain embodiments, an herbicide for killing or controlling the proliferation of weeds and other unwanted plants is combined with one of the active agents described above. Examples of herbicides include, but are not limited to, benzoic acid herbicides such as dicamba esters, phenoxyalkanoic acid herbicides such as 2,4-D, MCPA and 2,4-DB esters, aryloxyphenoxypropionic acid herbicides such as clodinafop, cyhalofop, fenoxaprop, fluazifop, haloxyfop and quizalofop esters, pyridinecarboxylic acid herbicides such as aminopyralid, picloram and clopyralid esters, pyrimidinecarboxylic acid herbicides such as aminocyclopyrachlor esters, pyridyloxyalkanoic acid herbicides such as fluoroxypyr and triclopyr esters, and hydroxybenzonitrile herbicides such as bromoxynil and ioxynil esters, esters of the arylpyridine carboxylic acids and arylpyrimidine carboxylic acids of the following generic structures as disclosed in U.S. Pat. No. 7,314,849, U.S. Pat. No. 7,300,907 and U.S. Pat. No. 7,642,220. In certain embodiments, the herbicide is selected from the group consisting of 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, amitrole, asulam, atrazine, azafenidin, benefin, bensulfuron, bensulide, bentazon, bromacil,

bromoxynil, butylate, carfentrazone, chloramben, chlorimuron, chlorproham, chlorsulfuron, clethodim, clomazone, clopyralid, cloransulam, cyanazine, cycloate, DCPA, desmedipham, dichlobenil, diclofop, diclosulam, diethatyl, difenzoquat, diflufenzopyr, dimethenamid-p, diquat, diuron, DSMA, endothall, EPTC, ethalfluralin, ethametsulfuron, ethofumesate, fenoxaprop, fluazifop-P, flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, fluthiacet, fomesafen, foramsulfuron, glufosinate, glyphosate, halosulfuron, haloxyfop, hexazinone, imazamethabenz, imazamox, imazapic, imazaquin, imazethapyr, isoxaben, isoxaflutole, lactofen, linuron, MCPA, MCPB, mesotrione, methazole, metolachlor-s, metribuzin, metsulfuron, molinate, MSMA, napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxasulfuron, oxyfluorfen, paraquat, pebulate, pelargonic acid, pendimethalin, phenmedipham, picloram, primisulfuron, prodiamine, prometryn, pronamide, propachlor, propanil, prosulfuron, pyrazon, pyridate, pyrithiobac, quinclorac, quizalofop, rimsulfuron, sethoxydim, siduron, simazine,

sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, terbacil, thiazopyr, thifensulfuron, thiobencarb, tralkoxydim, triallate, triasulfuron, tribenuron, triclopyr, trifluralin,

triflusulfuron, vernolate, and any combination thereof.

Fungicides

[0247] In certain embodiments, a fungicide for killing or controlling the proliferation of a fungus is combined with one of the active agents described above. Exemplary fungicides include, but are not limited to, strobilurins, azoxystrobin, dimoxystrobin, enestroburin, fluoxastrobin, kresoxim-methyl, metominostrobin, picoxystrobin,pyraclostrobin,

trifloxystrobin, orysastrobin, carboxamides, carboxanilides, benalaxyl, benalaxyl-M, benodanil, carboxin, mebenil, mepronil, fenfuram, fenhexamid, flutolanil, furalaxyl, furcarbanil, furametpyr, metalaxyl, metalaxyl-M (mefenoxam), methfuroxam, metsulfovax, ofurace, oxadixyl, oxycarboxin, penthiopyrad, pyracarbolid, salicylanilide, tecloftalam, thifluzamide, tiadinil, N-biphenylamides, bixafen, boscalid, carboxylic acid morpholides, dimethomorph, flumorph, benzamides, flumetover, fluopicolid (picobenzamid), zoxamid, carboxamides, carpropamid, diclocymet, mandipropamid, silthiofam, azoles, triazoles, bitertanol, bromuconazole, cyproconazole, difenoconazole, diniconazole, enilconazole, epoxiconazole, fenbuconazole, flusilazol, fluquinconazole, flutriafol, hexaconazole, imibenconazole, ipconazole, metconazole, myclobutanil, penconazole, propiconazole, prothioconazole, simeconazole, tebuconazole, tetraconazole, triadimenol, triadimefon, triticonazole, Imidazoles, cyazofamid, imazalil, pefurazoate, prochloraz, triflumizole, benzimidazoles, benomyl, carbendazim, fuberidazole, thiabendazole, ethaboxam, etridiazole, hymexazol, nitrogen-containing heterocyclyl compounds, pyridines, fuazinam, pyrifenox, pyrimidines, bupirimate, cyprodinil, ferimzone, fenarimol, mepanipyrim, nuarimol, pyrimethanil, piperazines, triforine, pyrroles, fludioxonil, fenpiclonil, morpholines, aldimorph, dodemorph, fenpropimorph, tridemorph, dicarboximides, iprodione,

procymidone, vinclozolin, acibenzolar-S-methyl, anilazine, captan, captafol, dazomet, diclomezin, fenoxanil, folpet, fenpropidin, famoxadon, fenamidon, octhilinone, probenazole, proquinazid, pyroquilon, quinoxyfen, tricyclazole, carbamates, dithiocarbamates, ferbam, mancozeb, maneb, metiram, metam, propineb, thiram, zineb, ziram, diethofencarb, flubenthiavalicarb, iprovalicarb, propamocarb, guanidines, dodine, iminoctadine, guazatine, kasugamycin, polyoxins, streptomycin, validamycin A, organometallic compounds, fentin salts, sulfur-containing heterocyclyl compounds, isoprothiolane, dithianone,

organophosphorous compounds, edifenphos, fosetyl, fosetyl-aluminum, iprobenfos, pyrazophos, tolclofos-methyl, Organochlorine compounds, thiophanate-methyl,

chlorothalonil, dichlofluanid, tolylfluanid, flusulfamide, phthalide, hexachlorobenzene, pencycuron, quintozene, nitrophenyl derivatives, binapacryl, dinocap, dinobuton,

spiroxamine, cyflufenamid, cymoxanil, metrafenon, N-2-cyanophenyl-3,4-dichloroisothiazol-

5- carboxamide (isotianil), N-(3',4',5'-trifluorobiphenyl-2-yl)-3-difluoromethyl- 1- methylpyrazole-4-carboxamide, 3-[5-(4-chlorophenyl)-2,3-dimethylisoxazolidin-3-yl]- pyridine, N-(3',4'-dichloro-4-fluorobiphenyl-2-yl)-3-difluoromethyl- 1-methylpyrazol- e-4- carboxamide, 5-chloro-7-(4-methylpiperidin-l-yl)-6-(2,4,6-trifluorophenyl)-[l,2,4]tria- zolo[l,5-a]pyrimidine, 2-butoxy-6-iodo-3-propylchromen-4-one, N,N-dimethyl-3-(3-bromo-

6- fluoro-2-methylindole-l-sulfonyl)-[l,2,4]triazo- le-l-sulfonamide, methyl-(2-chloro-5-[l- (3-methylbenzyloxyimino)-ethyl]benzyl)carbamate, methyl-(2-chloro-5-[l-(6-methylpyridin- 2-ylmethoxy-imino)ethyl]benzyl)carbamate, methyl 3-(4-chlorophenyl)-3-(2- isopropoxycarbonylamino-3-methylbutyryl-amino)propionate, 4-fluorophenyl N-(l-(l-(4- cyanophenyl)ethanesulfonyl)but-2-yl)carbamate , N-(2-(4-[3-(4-chlorophenyl)prop-2- ynyloxy]-3-methoxyphenyl)ethyl)-2-metha- nesulfonylamino-3-methylbutyramide, N-(2-(4- [3-(4-chlorophenyl)prop-2-ynyloxy]-3-methoxyphenyl)ethyl)-2-ethan- esulfonylamino-3- methylbutyramide, N-(4'-bromobiphenyl-2-yl)-4-difluoromethyl-2-methylthiazol-5- carboxamide, N-(4'-trifluoromethylbiphenyl-2-yl)-4-difluoromethyl-2-methylthiazol-5- carboxamide, N-(4'-chloro-3'-fluorobiphenyl-2-yl)-4-difluoromethyl-2-methylt- hiazol-5- carboxamide, and methyl 2-(ortho-((2,5-dimethylphenyloxy-methylene)phenyl)-3- methoxyacrylate.

Modulation of Traits in Plants, Insects and Plant Pathogens Plants

[0248] In another aspect, the present disclosure provides for a method of modulating a trait of a plant, comprising delivering to the plant an effective amount of a modified polyethyleneimine-based formulation comprising an oligonucleotide or polynucleotide that modulates the expression of a gene in the plant. Oligonucleotides or polynucleotides that modulate the expression of a gene in a plant include, but are not limited to, RNA molecules (e.g., siRNA, aiRNA, miRNA, dsRNA, and shRNA) and DNA molecules (e.g. , antisense polynucleotides) that decrease expression of the gene in the plant, and RNA molecules (e.g. , mRNA) and DNA molecules (e.g. , expression cassettes and plasmids) that increase expression of the gene in the plant. In certain embodiments, the oligonucleotide or polynucleotide modulates the expression of a gene that is endogenous to the plant. In other embodiments, the oligonucleotide or polynucleotide modulates the expression of a gene that is heterologous to the plant, e.g., a transgene that does not naturally occur within the plant. In certain embodiments, the oligonucleotide or polynucleotide that modulates the expression of a gene in the plant hybridizes to a gene or gene product that is endogenous to the plant.

[0249] Traits that may be modulated in a plant include, but are not limited to, total seed germination, rate of seed germination, disease tolerance, insect tolerance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, tolerance to heavy metals, total yield, seed yield, fruit yield, root growth, early vigor, plant growth, plant biomass, plant size, plant lifespan, total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number, leaf number, fruit size, fruit freshness, fruit ripening time, fruit nutritional content, plant nutritional content, and any combination thereof. In a particular embodiment, the presently disclosed modified polyethylenimine-based formulations can be used to deliver an active agent to a plant (e.g., a weed), for the purpose of killing and/or controlling the proliferation of the plant.

[0250] In certain embodiments, one or more of the above-mentioned traits in a plant is increased or improved relative to a plant that is not treated with the modified

polyethyleneimine-based formulation. The trait in the plant as described herein may be increased by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% by delivery of the modified polyethyleneimine-based formulation to the plant relative to a plant that is not treated with the formulation. In other embodiments, one or more of the above mentioned traits is decreased relative to a plant that is not treated with the modified polyethyleneimine-based formulation. The trait in the plant as described herein may be decreased by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% by delivery of the modified polyethyleneimine-based formulation to the plant relative to a plant that is not treated with the formulation. Insects

[0251] In another aspect, the present disclosure provides for a method of modulating a trait of an insect, comprising delivering to the insect, to a plant infested with the insect, or to a plant prior to infestation with the insect an effective amount of a modified

polyethyleneimine-based formulation comprising an oligonucleotide or polynucleotide that modulates expression of a gene in the insect. Oligonucleotides or polynucleotides that modulate the expression of a gene in the insect include, but are not limited to, RNA molecules (e.g. , siRNA, aiRNA, miRNA, dsRNA, and shRNA) and DNA molecules (e.g. , antisense polynucleotides) that decrease expression of the gene in the insect, and RNA molecules (e.g. , mRNA) and DNA molecules (e.g., expression cassettes and plasmids) that increase expression of the gene in the insect. In certain embodiments, the oligonucleotide or polynucleotide that modulates the expression of a gene in the insect hybridizes to a gene or gene product that is endogenous to the insect.

[0252] Traits that may be modulated in the insect include, but are not limited to, insect growth, development, activity, and/or lifespan. For example, in certain embodiments, delivery of the formulation to the insect kills the insect. In certain embodiments, delivery of the formulation to the insect reduces its growth and/or lifespan, thereby reducing the damage done by the insect to a plant. In certain embodiments, delivery of the formulation to the insect causes the insect to remain in a young or immature stage, thus preventing the insect from completing its lifecycle. For example, in certain embodiments, delivery of the formulation to the insect interferes with enzymes involved in the molting process that stimulate the synthesis and formation of chitin, which is an essential component of an insect's exoskeleton. As a result, the insect fails to reach adulthood because it dies in an immature stage. In certain embodiments, delivery of the formulation to the insect disrupts the feeding activity of the insect. As a result, insects starve to death because they are unable to obtain nutrients.

[0253] In certain embodiments, the delivery of the formulation to the insect decreases its growth, activity or lifespan by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% relative to an insect that is not treated with the

formulation. In certain embodiments, the delivery of the formulation to the insect increases its growth, activity or lifespan by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000% relative to an insect that is not treated with the formulation.

Plant Pathogens

[0254] In another aspect, the present disclosure provides a method of modulating the pathogenicity of a plant pathogen, comprising applying to the plant pathogen, to a plant infected with the plant pathogen, or to a plant prior to infection with the plant pathogen a modified polyethyleneimine-based formulation comprising an oligonucleotide or

polynucleotide that modulates expression of a gene in the plant pathogen. For example, in certain embodiments, the pathogenicity of the plant pathogen is decreased, for example by decreasing the growth, activity, or lifespan of the plant pathogen, or delaying the

development of the plant pathogen. In a particular embodiment, the formulation is used to kill the plant pathogen and/or control its proliferation. In certain other embodiments, the pathogenicity of the plant pathogen is increased, for example, by increasing the growth, activity or lifespan of the plant pathogen, or accelerating its development. Increasing pathogenicity of a plant pathogen may be used, for example, to kill or reduce the growth of a plant such as a weed. In certain embodiments, the growth, activity or lifespan of the plant pathogen may be decreased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% relative to a plant pathogen that is not treated with the modified polyethyleneimine-based formulation. In certain embodiments, the growth, activity or lifespan of the plant pathogen may be increased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000% relative to a plant pathogen that is not treated with the modified polyethyleneimine-based formulation.

Target Organisms

[0255] In certain embodiments, the target organism is any organism in which one or more traits is modulated by the active agent. In certain embodiments, a target organism is also a host organism, as described herein below. For example, in some embodiments, the target organism is an organism comprising one or more genes that is targeted by an oligonucleotide or polynucleotide active agent. In some embodiments, the target organism is a plant in which one or more yield-related traits is improved by the active agent. In some embodiments, the target organism is a beneficial insect whose growth, fecundity, or disease resistance is improved by the active agent. In certain embodiments, the target organisms are plant pests or pathogens whose damage to the plant can be reduced or eliminated by active agents according to the invention. Examples of plant pests and pathogens include, but are not limited to, insects, nematodes, fungi, bacteria, viruses, and parasitic plants such as striga, dodder, and mistletoe. Insect pests that may be targeted according to the invention include, but are not limited to, chewing, sucking, and boring insects that belong, for example, to the non-limiting Orders Coleoptera, Diptera, Hemiptera, Heteroptera, Homoptera, Hymenoptera, Lepidoptera, and Orthoptera.

Insects

[0256] In some embodiments the composition may be taken up by an insect by direct contact with the composition, for example, by topical adsorption or inhalation of the composition or by direct feeding on a bait comprising the composition, as described below. The compositions may also be taken up by the insect by direct feeding on a plant that has been treated with the composition. Examples of insect pests that may be targeted by the invention include, but are not limited to, those provided in Table 2.

[0257] Table 2.

Weeds

[0266] In certain embodiments, the target organism is a weed. As used herein, the term "weed" refers to any unwanted plant. The weed to be controlled may include monocotyledonous species, such as species of the genus Agrostis, Alopecurus, Avena, Bromus, Cyperus, Digitaria, Echinochloa, Lolium, Monochoria, Rottboellia, Sagittaria, Scirpus, Setaria, Sida or Sorghum, and dicotyledonous species, for example species of the genus Abutilon, Amaranthus, Chenopodium, Chrysanthemum, Conyza, Galium, Ipomoea, Nasturtium, Sinapis, Solarium, Stellaria, Veronica, Viola or Xanthium. Weeds can also include plants which may be considered crop plants but which are growing outside a crop area (escapes), or which grow from seed left over from a previous planting of a different crop (volunteers). Such volunteers or escapes may be tolerant to certain other herbicides.

[0267] It has been demonstrated that several agriculturally relevant traits in plants can be modified via the introduction of transgenes that target the silencing of specific genes, including soybean oil composition and corn kernel protein composition. dsRNAs targeting specific genes in specific species can be applied topically to alter plant traits as well, and in some cases, offers the farmer more flexibility with regard to timing and endurance of application. In certain embodiments the compositions of the present invention may be used to enhance a yield-related trait in a plant. Yield-related traits that may be enhanced by the compositions of the present invention include, but are not limited to, total seed germination, rate of seed germination, plant biomass, disease tolerance, insect tolerance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, tolerance to heavy metals, total yield, seed yield, root growth, early vigor, plant biomass, plant size, total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number, and leaf number.

Crop Plants

[0268] Representative crop plants that may be target organisms include

monocotyledonous and dicotyledonous plants including but not limited to fodder or forage legumes, ornamental plants, food crops, trees, or shrubs selected from Acer spp., Allium spp., Amaranthus spp., Ananas comosus, Apium graveolens, Arachis spp, Asparagus officinalis, Beta vulgaris, Brassica spp. (e.g., Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Camellia sinensis, Canna indica, Cannabis saliva, Capsicum spp., Castanea spp., Cichorium endivia, Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Coriandrum sativum, Corylus spp., Crataegus spp., Cucurbita spp., Cucumis spp., Daucus carota, Fagus spp., Ficus carica, Fragaria spp., Ginkgo biloba, Glycine spp. (e.g., Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g., Helianthus annuus), Hibiscus spp., Hordeum spp. (e.g., Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Linum usitatissimum, Litchi chinensis, Lotus spp., Lujfa acutangula, Lupinus spp., Lycopersicon spp. (e.g., Lycopersicon esculenturn, Lycopersicon lycopersicum, Lycopersicon pyriforme), Malus spp., Medicago sativa, Mentha spp., Miscanthus sinensis, Moms nigra, Musa spp., Nicotiana spp., Olea spp., Oryza spp. (e.g., Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Petroselinum crispum, Phaseolus spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prunus spp., Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis spp., Solanum spp. (e.g., Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Sorghum halepense, Spinacia spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticum spp. (e.g., Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare), Vaccinium spp., Vz^'cz^'a spp., Vigna spp., Vzo/a odorata, Vitis spp., and Zea rni s. Especially preferred are rice, oilseed rape, canola, soybean, corn (maize), cotton, sugarcane, alfalfa, sorghum, and wheat.

[0269] In certain embodiments, a target gene of interest may also include a gene that is essential to the survival of an organism, such as a weed, insect, or plant pathogen, and can serve as a target for controlling growth and proliferation of the organism. For example, dsRNA-mediated silencing of an essential gene in an insect pest can induce cessation of feeding and ultimately growth inhibition, morbidity, or mortality. Recent studies have shown that certain coleopteran insect species, most notably the western corn rootworm, Diabrotica virgifera virgifera, are exquisitely sensitive to ingested dsRNAs. Highly efficacious dsRNAs yield LC50 values in the parts-per-billion (ppb or ng/ml) range with this species. RNAi provides a unique mode of action for the control of insect pests that could complement the current strategy of expressing Bacillus thuringiensis insecticidal proteins in plants of agricultural importance. In certain embodimetnts, dsRNAs targeting essential insect genes can be delivered via topical sprays for RNAi-mediated insect control.

Non-Target Organisms

[0270] In some embodiments, the compositions of the invention may be applied to an organism that is different from the target organism. For example, in some embodiments the target organism is an insect, and the composition is applied to a non-target organism, such as a plant, that is a host for the insect. As used herein, a "non-target organism" is any organism other than the target organism. Where the target organism and host organism differ, a non- target organism can comprise a host organism and organisms that consume the host organism or otherwise contact polynucleotides (e.g., siRNAs or antisense polynucleotides) or proteins expressed in a host organism. The target- specific design of polynucleotides such as RNAi and antisense polynucleotides, as described herein, provides that such polynucleotides have little or no gene silencing activity in non-target organisms.

[0271] Non-target organisms include crop plants that may be infected with a target organism, such as a plant pathogen or insect. Representative crop plants include

monocotyledonous and dicotyledonous plants including but not limited to fodder or forage legumes, ornamental plants, food crops, trees, or shrubs selected from Acer spp., Allium spp., Amaranthus spp., Ananas comosus, Apium graveolens, Arachis spp, Asparagus officinalis, Beta vulgaris, Brassica spp. (e.g., Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Camellia sinensis, Canna indica, Cannabis saliva, Capsicum spp., Castanea spp., Cichorium endivia, Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Coriandrum sativum, Corylus spp., Crataegus spp., Cucurbita spp., Cucumis spp., Daucus carota, Fagus spp., Ficus carica, Fragaria spp., Ginkgo biloba, Glycine spp. (e.g., Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g., Helianthus annuus), Hibiscus spp., Hordeum spp. (e.g., Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Lycopersicon spp. (e.g., Lycopersicon esculenturn, Lycopersicon lycopersicum, Lycopersicon pyriforme), Malus spp., Medicago sativa, Mentha spp., Miscanthus sinensis, Moms nigra, Musa spp., Nicotiana spp., Olea spp., Oryza spp. (e.g., Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Petroselinum crispum, Phaseolus spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prunus spp., Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis spp., Solanum spp. (e.g., Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Sorghum halepense, Spinacia spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticum spp. (e.g., Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare), Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., and Zea mays. Especially preferred are rice, oilseed rape, canola, soybean, corn (maize), cotton, sugarcane, alfalfa, sorghum, and wheat. Application of the Formulations

[0272] In certain embodiments, the presently disclosed formulations can be applied as a spray or powder to the plant, plant part, seed, a pest, or an area of cultivation. The presently disclosed formulations may also be applied as concentrated emulsions, dusts, emulsifiable concentrates, fumigants, gels, granules, seed treatments, suspension concentrates, suspoemulsions, tablets, water soluble liquids, water dispersible granules or dry flowables, wettable powders, and ultra-low volume solutions. For further information on formulation types see "Catalogue of Pesticide Formulation Types and International Coding System" Technical Monograph No. 2, 5th Edition by CropLife International (2002), which is incorporated herein by reference in its entirety. Agricultural formulations are also described, for example, in U.S. Pat. No. 8,815,271, which is incorporated herein by reference in its entirety.

[0273] For example, the presently disclosed formulations may be applied as aqueous suspensions or emulsions prepared from concentrated formulations. Such water-soluble, water-suspendable, or emulsifiable formulations can either be solids, usually known as wettable powders, or water dispersible granules, or liquids usually known as emulsifiable concentrates, or aqueous suspensions. Wettable powders, which may be compacted to form water dispersible granules, comprise an intimate mixture of the composition, a carrier, and surfactants. The carrier may be selected from attapulgite clays, montmorillonite clays, diatomaceous earths, and purified silicates. Effective surfactants, comprising from about 0.5% to about 10% of the wettable powder, include sulfonated lignins, condensed

naphthalenesulfonates, naphthalenesulfonates, alkylbenzenesulfonates, alkyl sulfates, and non-ionic surfactants such as ethylene oxide adducts of alkyl phenols.

[0274] Emulsifiable concentrates can comprise a suitable concentration of the presently disclosed formulation, such as from about 50 to about 500 grams per liter of liquid dissolved in a carrier that is either a water-miscible solvent or a mixture of water-immiscible organic solvent and emulsifiers. Suitable organic solvents include aromatics, especially xylenes and petroleum fractions, especially the high-boiling naphthalenic and olefinic portions of petroleum such as heavy aromatic naphtha. Other organic solvents may also be used, such as the terpenic solvents including rosin derivatives, aliphatic ketones such as cyclohexanone, and complex alcohols such as 2-ethoxyethanol. Suitable emulsifiers for emulsifiable concentrates can be selected from conventional anionic and non- ionic surfactants.

[0275] Aqueous suspensions comprise suspensions of water-insoluble forms of the presently disclosed formulations dispersed in an aqueous carrier at a concentration in the range from about 5% to about 50% by weight. Ingredients, such as inorganic salts and synthetic or natural gums, may also be added to increase the density and viscosity of the aqueous carrier.

[0276] The presently disclosed formulations may also be applied as granular formulations, for example, for applications to the soil. Granular formulations may contain from about 0.5% to about 10% by weight of the composition, dispersed in a carrier that comprises clay or a similar substance. Such formulations may be prepared by dissolving the formulation in a suitable solvent and applying it to a granular carrier which has been preformed to a suitable particle size, for example, in the range of from about 0.5 to about 3 mm. Such formulations may also be prepared by making a dough or paste of the carrier and compound and crushing and drying to obtain the desired granular particle size.

[0277] Dusts comprising the presently disclosed formulations may be prepared by intimately mixing the formulation in powdered form with a suitable dusty agricultural carrier, such as kaolin clay, ground volcanic rock, and the like. Dusts may contain from about 1% to about 10% by weight of the formulation. They may be applied as a seed dressing or as a foliage application with a dust blower machine.

[0278] The presently disclosed formulations may also be applied in the form of a solution in an appropriate organic solvent (e.g., petroleum oil) such as the spray oils, which are widely used in agricultural chemistry.

[0279] The presently disclosed formulations may also be applied in the form of an aerosol composition. The formulation can be dissolved or dispersed in a carrier, which is a pressure-generating propellant mixture. The aerosol composition is packaged in a container from which the mixture is dispensed through an atomizing valve.

[0280] The presently disclosed formulations may be applied to the crop area or plant to be treated, simultaneously or in succession with further compounds. These further compounds can be, for example, fertilizers or micronutrient donors or other preparations, which influence the growth of plants. They can also be selective herbicides or no n- selective herbicides as well as insecticides, fungicides, bactericides, nematicides, or mixtures of several of these preparations, if desired together with further carriers, surfactants or application promoting adjuvants customarily employed in the art of formulation.

Therapeutic Applications

[0281] In another aspect, the presently disclosed modified PEI-based formulations can be used to deliver a therapeutic agent to a target in a subject for the purpose of treating or preventing a disease or disorder. In certain embodiments, the presently disclosed modified PEI-based formulations are combined with a pharmaceutically acceptable excipient and/or carrier to form a pharmaceutical formulation. In certain embodiments, the disease or disorder is treated or prevented by administering a therapeutically effective amount of the

pharmaceutical formulation to a subject in need thereof. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0282] In certain embodiments, the presently disclosed modified PEI-based formulations, i.e., complexes, microparticles, nanoparticles, picoparticles, liposomes, and micelles, are combined with one or more pharmaceutically acceptable excipients and/or carriers to form pharmaceutical formulations suitable to administer to mammals, including humans. Examples of classes of such excipients and carriers include, but are not limited to, fillers, extenders, binders, humectants, disintegrants, plasticizers, stabilizers, solution retarding agents, wetting agents, suspending agents, thickening agents, absorbents, lubricants, surfactants, buffering agents, diluents, solvents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, opacifying agents, separating agents, and coating permeability adjusters. Excipients and/or carriers may comprise about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or higher percentage of the presently disclosed pharmaceutical formulations.

[0283] The presently disclosed pharmaceutical formulations can be administered to mammals, including humans, by any conventional route. Examples of such routes include, but are not limited to, orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically, bucally, or as an oral or nasal spray. Dosage forms for oral administration include, but are not limited to, solid and liquid dosage forms. Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, pills, powders, and granules. Liquid dosage forms for oral administration include, but are not limited to, emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. Dosage forms for topical administration include, but are not limited to, ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and transdermal patches.

[0284] As used herein, "parenteral administration" and "administered parenterally" means modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0285] Although the dosage will vary depending on the symptoms, age and body weight of the subject, the nature and severity of the disorder to be treated or prevented, the route of administration and the form of the drug, in general, a daily dosage of from 0.01 to 2000 mg of the therapeutic agent is recommended for an adult human patient, and this may be administered in a single dose or in divided doses.

[0286] As used herein, the term "therapeutic agent" includes any synthetic or naturally occurring biologically active compound or composition which, when administered to subject, induces a desired pharmacologic, immunogenic, and/or physiologic effect by local and/or systemic action. The term therefore encompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals, including molecules such as proteins, peptides, hormones, nucleic acids, and gene constructs. More particularly, the term "therapeutic agent" includes compounds or compositions for use in all of the major therapeutic areas.

[0287] Examples of such therapeutic agents include, but are not limited to, nucleic acids, adjuvants, anti-infectives such as antibiotics and antiviral agents, analgesics and analgesic combinations, anorexics, anti- inflammatory agents, anti-epileptics, local and general anesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics,

antiarrhythmics, antihypertensive agents, hormones, and nutrients, antiarthritics,

antiasthmatic agents, anticonvulsants, antihistamines, antinauseants, antineoplastics, antipruritics, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrythmics), antihypertensives, diuretics, vasodilators, central nervous system stimulants, cough and cold preparations, decongestants, diagnostics, hormones, bone growth stimulants and bone resorption inhibitors, oncology drugs (e.g., chemotherapy drugs, hormonal therapeutic agents, immunotherapeutic agents, radiotherapeutic agents), lipid-lowering agents, antidepressants, stimulants, antibiotics, birth control medication, anti-angiogenics, cytovascular agents, signal

transduction inhibitors, hormones, vasoconstrictors, and steroids, immunosuppressives, muscle relaxants, psychostimulants, sedatives, tranquilizers, proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced), small molecules and other biologically active macromolecules such as, for example, proteins and enzymes. The agent may be a biologically active agent used in medical, including veterinary, applications. The term therapeutic agent also includes without limitation, medicaments, vitamins; mineral supplements, substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness, substances which affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment. These therapeutic agents may be administered alone with pharmaceutical formulations or in combination (e.g. , co -administered) with pharmaceutical formulations comprising nucleic acid, such as interfering RNA.

[0288] Examples of nucleic acids include, but are not limited to, interfering RNA molecules (e.g. , siRNA, aiRNA, miRNA), antisense oligonucleotides, plasmids, ribozymes, immuno stimulatory oligonucleotides, and mixtures thereof.

[0289] Nucleic acids delivered as the therapeutic agent in the presently disclosed pharmaceutical formulations can be used to downregulate or silence the translation (i.e. , expression) of a target gene of interest in the target organism. Examples of classes of genes that can be downregulated or silenced include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g. , liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenic genes, immunomodulator genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders. Examples viral sequences that can be downregulated or silenced include filo viruses such as Ebola virus and Marburg virus, arenaviruses such as Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus, influenza viruses such as Influenza A, B, and C viruses, hepatitis viruses, Human Immunodeficiency Virus (HIV), herpes viruses, and Human Papilloma Viruses (HPV).

[0290] Examples of peptides or polypeptide that may be used as therapeutic agents include, but are not limited to, an antibodies such as a polyclonal antibodies, a monoclonal antibodies, antibody fragments, humanized antibodies, recombinant antibodies, recombinant human antibodies, Primatized™ antibodies, cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell-surface receptors, ligands, hormones, or small molecules.

[0291] Examples of oncology drugs that may be used as therapeutic agents include, but are not limited to, alkeran, allopurinol, altretamine, amifostine, anastrozole, araC, arsenic trioxide, bexarotene, biCNU, carmustine, CCNU, celecoxib, cladribine, cyclosporin A, cytosine arabinoside, Cytoxan, dexrazoxane, DTIC, estramustine, exemestane, FK506, gemtuzumab-ozogamicin, hydrea, hydroxyurea, idarubicin, interferon, letrozole, Leustatin, leuprolide, litretinoin, megastrol, L-PAM, mesna, methoxsalen, mithramycin, nitrogen mustard, pamidronate, Pegademase, pentostatin, porfimer sodium, prednisone, rituxan, streptozocin, STI-571, taxotere, temozolamide, VM-26, toremifene, tretinoin, ATRA, valrubicin, velban, ellipticin and ellipticin analogs or derivatives, epothilones, intracellular kinase inhibitors, and camptothecins.

[0292] Examples of anti-viral drugs that may be used as therapeutic agents include, but are not limited to, abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fixed dose combinations, fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion inhibitors, ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, interferon type III (e.g., IFN-λ molecules such as IFN-λΙ, IFN- 2, and ΙΡΝ-λ3), interferon type II (e.g., IFN-γ), interferon type I (e.g., IFN-a such as PEGylated IFN-a, IFN-β, IFN-K, IFN-δ, IFN-ε, IFN-τ, IFN-co, and IFN-ζ, interferon, lamivudine, lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, synergistic enhancers, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine.

[0293] In certain embodiments, the therapeutic agent to be delivered may be a prophylactic agent. Prophylactic agents include, but are not limited to, antibiotics, nutritional supplements, and vaccines. Vaccines may comprise isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, and cell extracts. Prophylactic agents may be combined with interleukins, interferon, cytokines, and adjuvants such as cholera toxin, alum, and Freund's adjuvant.

[0294] Examples of prophylactic agents include, but are not limited to, (1) antigens of the following bacterial organisms: Streptococccus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,

Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans, Borrelia burgdorferi, and Camphylobacter jejuni, (2) antigens of the following viruses: smallpox, influenza A and B, respiratory syncytial virus, parainfluenza, measles, HIV, varicella- zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhino virus, adenovirus, papillomavirus, polio virus, mumps, rabies, rubella, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and hepatitis A, B, C, D, and E virus, and (3) antigens of the following fungal, protozoan, and parasitic organisms: Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma

pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum,

Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni. These antigens may be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof.

[0295] The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

EXAMPLES

[0296] Example 1 - Synthesis of 2-Tridecyloxirane.

[0297] At room temperature, 6.31 g of pentadec-l-ene (30.00 mmol) was added to 750 mL of acetonitrile in a 1 liter 3-neck round bottom flask, equipped with a stir bar. To this was added 309 mg of cobalt(II) perchlorate hexahydrate (1 mmol) in a single portion, washing in with a small amount of acetonitrile. To this solution was added 13.44 mg of 3- chloroperbenzoic acid (60.00 mmol) portionwise over about 2 minutes. Little, if any, exotherm was noted, but a greenish color develops over time. The reaction was stirred for 1 hour at room temperature and then filtered through a pad of silica gel. The pad was washed with a few portions of acetonitrile and then the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified via MP-HPLC on a 100 g silica cartridge, eluting using a solvent mixture of 25% ethyl acetate in hexanes.

Appropriate fractions were combined and solvent was removed under reduced pressure to obtain the desired product as an oil (5.9 g; 87% theory).

[0298] Example 2 - Synthesis of Reference Compound (7C1) - General Synthesis

Method for Epoxidation of Poly(ethyleneimines).

[0299] A 2 dram reaction vial was equipped with a stir bar. To this vial was added

192 mg of poly(ethylenimine) (MW600) (4.45 mmol nominally), followed by 1.43 mL of 2- tridecyloxirane (4.45 mmol). Approximately 2 mL of ethanol was then added, resulting in a two-phase mixture. The charged reaction vial was then heated to 90 C for 72 hours, resulting in a homogeneous solution. Ethanol was removed under reduced pressure to afford the requisite material. In some cases, the residue was pre-absorbed onto silica and purified on a 50 g column, eluted using a solvent gradient of from 0 to 20% methanol in

dichloromethane (with 10% ammonium hydroxide in the methanol).

[0300] The same general reaction methodology was also utilized for the Michael addition of acrylates and acrylamides to poly(ethyleneimines). [0301] Example 3 - Synthesis of 13-(Oxiran-2-ylmethyl)- l,4,7,10-tetraoxa-13- azacyclopentadecane.

[0302] To a 50 mL 3-neck round bottom flask, 1.97 g of 1,4,7, 10-tetraoxa-13- azacyclopentadecane (9.00 mmol) was added to 10.62 g of neat 2-(chloromethyl)oxirane (114.75 mmol). To this was added approximately 0.4 mL of water and the reaction mixture was stirred at 40 C for 90 minutes. The reaction was then cooled back to ambient and 1.80 g of sodium hydroxide (50% aqueous solution; 22.50 mmol) was added in a single portion. The reaction mixture was then stirred at ambient temperature for another hour and then transferred to a separatory funnel. The aqueous layer was separated and then extracted three times with dichloro methane. The combined organic layer was washed with a small portion of saturated sodium chloride solution and then dried over magnesium sulfate. Solvent was removed under reduced pressure to obtain the crude product as an oil. The crude product was purified by vacuum distillation to obtain the desired product as a clear oil. Both NMR and mass spectra were consistent with the desired product.

[0303] Example 4 - Synthesis of 2-[2-[2-[2-(2-

Methoxyethoxy)ethoxy]ethoxy]ethoxy-methyl]oxirane.

[0304] 1.36 g of tetrabutylammonium hydrogensulfate (4.00 mmol) was added to a

125 mL 3-neck round bottomed flask, followed by 24.15 g of 50% aqueous sodium hydroxide (302.00 mmol) and then cooled in an ice/water bath, causing a precipitate to form. 8.33 g of 2-[2-[2-(2-methoxyethoxy)-ethoxy]ethoxy]ethanol (40.00 mmol) was added in a single portion and this mixture was stirred for 10 minutes. 29.61 g of 2- (chloromethyl)oxirane (25 mL, 320.00 mmol) was placed into an addition funnel and added dropwise, maintaining the temperature around 3 C throughout the addition. During this time the precipitate disappeared. The addition required approximately 20 minutes and then the reaction was stirred cold for another 1 hour before the ice/water bath removed. The reaction was stirred overnight at ambient temperature and then was extracted four times with ether. The combined organic washings were washed twice with small portions of saturated sodium chloride solution and then dried over magnesium sulfate. Solvent was removed under reduced pressure and the resulting oil was purified by vacuum distillation. Both NMR and mass spectrum were consistent with the expected structure. [0305] Example 5 - Modified PEI / Active Agent Formulation

[0306] Distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-di-(9Z-octadecenoyl)- sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C14-PEG) were each dissolved in ethanol and mixed in a molar ratio of DSPC:DOPE:Cholesterol:C14-PEG of 3.4:3.4:26: 1. This ethanol mixture was then added to 10 mM citrate buffer (pH 5) while stirring to spontaneously form empty liposomes. siRNA and modified PEI were mixed at a weight ratio of 1 to 20. This mixture was then added to the empty liposomes solution and incubated at 37 °C for 0.5 hour. The formulation was then dialyzed against 10 mM citrate buffer (pH 5). This formulation yields a mean particle diameter of 217.2 nm with 99.6% entrapment efficiency.

[0307] Example 6 - Modified PEI / Active Agent Formulation

[0308] Modified PEI, l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-

(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C14-PEG) are dissolved in 90% ethanol in a molar ratio of 50: 10:38.5: 1.5. siRNA is dissolved in 10 mM citrate buffer (pH 5). These ethanolic and aqueous solutions are then mixed using micro fluidic-based mixing device (Precision Nanoassembler). The formulation is purified by 30K MWCO membrane spin column using 10 mM citrate buffer (pH 5).

[0309] Example 7 - Modified PEI / Active Agent Formulation

[0310] Modified PEI, (l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (C14-PEG) are each dissolved in absolute ethanol and mixed in a molar ratio of 50: 10:38.5: 1.5 (PEI:DSPC:Cholesterol:PEG). Citrate buffer is added to this ethanolic mixture for a final aqueous volume of 10 % v/v. This

aqueous/ethanolic mixture is added drop- wise to RNA dissolved in citrate buffer (pH 5). The resulting nanoparticle formulation is purified by passing it through a previously equilibrated PD-10 gel filtration column (Sephadex G-25, GE Healthcare) to remove unincorporated excipients. [0311] Example 8 - General Procedure for Polyplex Formulation of Active Agents with Modified PEIs

[0312] Modified PEI dissolved in citrate buffer (pH 3.0) is added to RNA dissolved in citrate buffer (pH 5.0) in 0.75, 1.5, 3, 5, 10, and 20 weight/weight ratios. The resulting polyplexes are used without further purification. These formulations yield an average particle diameter of -10 nm with 99% entrapment efficiency.

[0313] Example 9 - General Procedure for Polyplex Formulation of Active Agents with Modified PEIs and PEG

[0314] Modifed PEI dissolved in citrate buffer (pH 3.0) and 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000 (i.e. , PEG-5000) dissolved in citrate buffer (pH 5.0) is added to RNA dissolved in citrate buffer (pH 5.0) at 0.75, 1.5, 3, 5, 10, and 20 weight/weight ratio. The resulting polyplexes are used without further purification. These formulations yield an average particle diameter of -10 nm with 99% entrapment efficiency.

[0315] Example 10 - General Procedure for Evaluating Modified PEI Formulations is

Insect Feeding Assays

[0316] Modified PEI formulations of the present disclosure can be evaluated in insect feeding assays to determine their efficacy in RNA delivery to an insect cell. Two model insects are used: western tarnished plant bug (WTPB, Lygus hesperus) and tarnished plant bug (TPB, Lygus lineolaris). Each modified PEI of the present disclosure to be evaluated is separately formulated with an siRNA that targets an essential gene in TPB and an siRNA that targets an essential gene in WTPB according to the general procedure described above in Example 4. The feeding assay employed is based on a 96 well format and a sachet system as described by Habibi et al. (2002, Archives of Insect Biochem. and Phys. 50: 62-74) and U.S. Patent No. 8,609,936, each of which is incorporated herein by reference in their entireties. The insect artificial diet is commercially available from Bio-Serv™ (Bio-Serv™ Diet F9644B, Frenchtown, N.J.).

[0317] Autoclaved boiling water is combined with Bio-Serv^® Diet F9644B in a surface sterilized blender. Four surface sterilized chicken eggs are broken and the contents are added to the blender containing the diet mix. The mixture is blended until smooth and adjusted to one liter of volume and allowed to cool. Feeding samples are prepared by mixing the siRNA formulations described above in the desired concentration with an equivalent volume of the blended diet.

[0318] A sheet of Parafilm^® (Pechiney Plastic Packing, Chicago, 111.) is placed over a

96-well format vacuum manifold with a vacuum of approximately -20 millimeters mercury, which is sufficient to cause extrusion of the Parafilm^® into the wells. Forty microliters of test sample are added to the Parafilm^® wells. A sheet of Mylar film (Clear Lam Packaging, Inc., Elk Grove Village, 111.) is then placed over the Parafilm^® and sealed gently with a tacking iron (Bienfang Sealector II, Hunt Corporation, Philadelphia, Pa.). The Parafilm^® sachets are then placed over a flat-bottom 96-well plate containing the Lygus eggs suspended in agarose. Upon hatching, Lygus nymphs will feed by piercing the sachet that is presented above them. Insect diet sachets are replaced on days two and four. Stunting and mortality scores are determined on day 5 and compared to the untreated controls. Those siRNA formulations that significantly increase stunting and mortality relative to the untreated controls demonstrate that the formulations are effective in delivering the siRNAs to the insect cells.

Claims

1. A modified polyethyleneimine comprising a structural unit of formula (I) and/or formula (II):

wherein:

Ri is hydrogen, a group of formula (III):

wherein

R₅ is hydrogen, an optionally substituted aliphatic or

heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group; a group of formula (IV):

(IV) wherein

are, independently, hydrogen or an optionally substituted aliphatic group;

R9 is an optionally substituted aliphatic group; and

X is O or NR11, wherein Rn is selected from the group consisting

H and Ci to C10 alkyl; or a group of formula (V):

wherein

Rio is selected from the group consisting of Ci to C₄ alkyl groups;

are, independently, hydrogen, a group of formula (III):

wherein R5 and R₆ are as defined above; or a group of formula (IV):

wherein R₇, Rg, R9, and X are as defined above; wherein the nitrogen atom bonded to Ri in formula (I) and the nitrogen atom bonded R₄ in formula (II) can each, independently, instead be bonded to an ethylene moiety in the modified polyethyleneimine; and with the proviso that at least one of Ri, R₂, R₃, and R₄ is a group of formula (III) or a group of formula (IV).

2. The modified polyethyleneimine of claim 1, wherein at least one of Ri, R₂, R₃, and R₄ is a group of formula (III).

3. The modified polyethyleneimine of claim 2, wherein at least Ri and R₂ are each, independently, a group of formula (III).

4. The modified polyethyleneimine of claim 2, wherein at least Ri, R₂, and R₃ are each, independently, a group of formula (III).

5. The modified polyethyleneimine of claim 2, wherein Ri, R₂, R₃, and R₄ are each, independently, a group of formula (III).

6. The modified polyethyleneimine of any one of claims 2-5, wherein R₆ is a Ci to C₂o alkyl group.

7. The modified polyethyleneimine of claim 6, wherein R₆ is a C₄ to Ci₆ alkyl group. 8. The modified polyethyleneimine of claim 6, wherein R₆ is a C₆ to C₁₄ alkyl group. 9. The modified polyethyleneimine of claim 3, wherein R5 is H and R₆ is selected from the group consisting of groups of formulae (XI) through (XXVII):

10. The modified polyethyleneimine of claim 1, wherein at least one of Ri, R₂, R₃, and R₄s a group of formula (IV).

11. The modified polyethyleneimine of claim 10, wherein at least Ri and R₂ are each, independently, a group of formula (IV).

12. The modified polyethyleneimine of claim 10, wherein at least Ri, R₂, and R₃ are each, independently, a group of formula (IV).

13. The modified polyethyleneimine of claim 10, wherein Ri, R₂, R₃, and R₄ are each, independently, a group of formula (IV).

14. The modified polyethyleneimine of any one of claims 10- 13, wherein R₉ is a Ci to C₂o alkyl group.

15. The modified polyethyleneimine of claim 14, wherein R₉ is a C₈ to Ci₆ alkyl group.

16. The modified polyethyleneimine of claim 11, wherein R₇ and R₈ are each hydrogen,_, Xs O, and R₉ is a group selected from the group consisting of formulae (XXVIII),

(XXIX), and (XXX):

(XXX).

The modified polyethyleneimine of claim 11, wherein R₇ and Rg are each hydrogen, X is NH, and R9 is a group selected from the group consisting of formulae (XXIX) and (XXX):

(XXX).

The modified polyethyleneimine of claim 1, wherein the modified polyethyleneimine is branched.

19. The modified polyethyleneimine of claim 18, wherein the modified

polyethyleneimine is hyperbranched.

20. The modified polyethyleneimine of claim 18, wherein the modified

polyethyleneimine is dendritic.

21. The modified polyethyleneimine of claim 18, wherein the modified

polyethyleneimine is derived from a branched polyethyleneimine having a number average molecular weight (M_n) in the range of from 600 to 10,000 Daltons.

22. The modified polyethyleneimine of claim 18, wherein the branched

polyethyleneimine has a number average molecular weight (M_n) of greater than 2,000

Daltons.

23. The modified polyethyleneimine of claim 21, wherein the branched

polyethyleneimine has a number average molecular weight (M_n) selected from the group consisting of 600, 1,200, 1,800, and 10,000 Daltons.

24. The modified polyethyleneimine of claim 18, wherein the modified

polyethyleneimine is derived from a branched polyethyleneimine comprising primary, secondary, and tertiary amino groups present in a ratio of 1 :2: 1.

25. The modified polyethyleneimine of claim 1, wherein the modified polyethyleneimine is linear.

26. The modified polyethyleneimine of claim 25, wherein the modified

polyethyleneimine is derived from a linear polyethyleneimine having an average molecular weight (M_n) of greater than 2,000 Daltons.

27. The modified polyethyleneimine of claim 26, wherein the linear polyethyleneimine has a number average molecular weight (M_n) in the range of from 2,500 to 100,000 Daltons.

28. The modified polyethyleneimine of claim 27, wherein the polyethyleneimine has a number average molecular weight (M_n) of 2,500 Daltons.

29. The modified polyethyleneimine of claim 1, wherein the modified polyethyleneimine is derived from the partial hydrolysis of a poly(2-alkyl-2-oxazoline).

30. The modified polyethyleneimine of claim 29, wherein the poly(2-alkyl-2-oxazoline) is poly(2-ethyl-2-oxazoline).

31. The modified polyethyleneimine of claim 1, wherein Ri is a group of formulae (III), (IV), or (V), R₂ and/or R₃ is a group of formulae (III) or (IV), and R₄ is a group of formulae (III) or (IV).

32. The modified polyethyleneimine of claim 1, wherein Ri and R₄ are each a group of formulae (III) or (IV), and R₂ and/or R₃ is a group of formulae (III) or (IV).

33. The modified polyethyleneimine of claim 32, wherein Ri and R₄ are each a group of formulae (III) and R₂ and/or R₃ is a group of formulae (III).

34. The modified polyethyleneimine of claim 32, wherein Ri and R₄ are each a group of formulae (IV) and R₂ and/or R₃ is a group of formulae (IV).

35. The modified polyethyleneimine of claim 1, wherein each Ri and R₄ is independently selected from at least two different groups of formulae (III) and/or (IV), and each R₂ and/or R₃ is independently selected from at least two different groups of formulae (III) and/or (IV).

36. The modified polyethyleneimine of claim 35, wherein each Ri and R₄ is

independently selected from at least two different groups of formulae (III), and each R₂ and/or R₃ is independently selected from at least two different groups of formulae

(III) .

37. The modified polyethyleneimine of claim 35, wherein each Ri and R₄ is

independently selected from at least two different groups of formulae (IV), and each R₂ and/or R₃ is independently selected from at least two different groups of formulae

(IV) .

38. A modified polyethyleneimine selected from the group consisting of compounds of formulae (1) through (299), wherein:

M_n is the number average molecular weight of the unmodified

polyethyleneimine prior to modification;

L/B denotes whether the modified polyethyleneimine is linear or branched;

R is the ratio of modified to unmodified primary and secondary amino groups in the modified polyethyleneimine; and the structure of moiety or moieties with which the structure of the polyethyleneimine has been modified;

39. A microparticle or nanoparticle comprising the modified polyethyleneimine of claim 1 and an active agent to be delivered.

40. The microparticle or nanoparticle of claim 39, wherein the active agent to be

delivered is selected from the group consisting of polynucleotides, oligonucleotides, proteins, peptides, and small molecules.

41. The microparticle or nanoparticle of claim 40, wherein the active agent to be

delivered is an oligonucleotide or a polynucleotide.

42. The microparticle or nanoparticle of claim 41, wherein the oligonucleotide or

polynucleotide is modified.

43. The microparticle or nanoparticle of claim 41, wherein the oligonucleotide or

polynucleotide is unmodified.

44. The microparticle or nanoparticle of claim 41, wherein the active agent to be

delivered is an RNA.

45. The microparticle or nanoparticle of claim 44, wherein the RNA is a single- stranded RNA.

46. The microparticle or nanoparticle of claim 44, wherein the RNA is a double- stranded RNA.

47. The microparticle or nanoparticle of claim 44, wherein the RNA is a small interfering RNA (siRNA).

48. The microparticle or nanoparticle of claim 44, wherein the RNA is a messenger RNA (mRNA).

49. A formulation comprising the modified polyethyleneimine of claim 1 and a first active agent to be delivered.

50. The formulation of claim 49, wherein the first active agent to be delivered and at least one modified polyethyleneimine of claim 1 are non-covalently associated to one another to form a non-covalent complex.

51. The formulation of claim 50, further comprising at least one additional active agent to be delivered.

52. The formulation of claim 51, wherein the at least one additional active agent to be delivered is contained within or on the surface of the non-covalent complex.

53. The formulation of claim 51, wherein the at least one additional active agent to be delivered is not contained within or on the surface of the non-covalent complex.

54. The formulation of claims 49-53, further comprising one or more excipients.

55. The formulation of claim 54, wherein the one or more excipients is selected from the group consisting of fillers, extenders, binders, humectants, disintegrants, plasticizers, stabilizers, solution retarding agents, wetting agents, suspending agents, thickening agents, absorbents, lubricants, surfactants, buffering agents, diluents, solvents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, opacifying agents, separating agents, and coating permeability adjusters.

56. The formulation of claim 54, wherein the one or more excipients is selected from the group consisting of sterols, carbohydrates, proteins, lipids, water-soluble polymers, and any combination thereof.

57. The formulation of claim 56, wherein the one or more excipients comprises

cholesterol.

58. The formulation of claim 56, wherein the one or more excipients comprises a

polyethylene glycol, a polypropylene oxide, a polyvinylpyrrolidone, a polyvinyl alcohol, polylactic acid, poly(lactic-co-glycolic acid), or any combination thereof.

59. The formulation of claim 49, wherein the first active agent to be delivered is an

oligonucleotide or a polynucleotide.

60. The formulation of claim 59, further comprising an agriculturally acceptable carrier.

61. The formulation of claim 60, wherein the oligonucleotide or polynucleotide modulates the expression of a gene in a plant.

62. The formulation of claim 60, wherein the oligonucleotide or polynucleotide

modulates the expression of a gene in an insect.

63. The formulation of claim 60, wherein the oligonucleotide or polynucleotide

modulates the expression of a gene in a plant pathogen.

64. The formulation of claim 51, wherein the at least one additional active agent is

selected from the group consisting of an herbicide, an insecticide, a fungicide, a bactericide, a viricide, and any combination thereof.

65. A method of regulating expression of a gene in an organism, comprising applying the formulation of any one of claims 49-64 to the organism.

66. A method of modulating a trait of a plant, comprising delivering to the plant an

effective amount of the formulation of claim 61.

67. The method of claim 66, wherein the trait is selected from the group consisting of total seed germination, rate of seed germination, disease tolerance, insect tolerance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, tolerance to heavy metals, total yield, seed yield, fruit yield, root growth, early vigor, plant growth, plant biomass, plant size, plant lifespan, total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number, leaf number, fruit size, fruit freshness, fruit ripening time, fruit nutritional content, plant nutritional content, plant sensitivity to herbicide, and any combination thereof.

68. The method of claim 67, wherein one or more of the traits is improved relative to a plant not treated with the formulation.

69. The method of claim 68, wherein at least one trait selected from the group consisting of plant growth, plant lifespan, plant size, fruit size, fruit yield, total yield, fruit freshness, fruit ripening time, plant nutritional content, and fruit nutritional content, is improved relative to a plant not treated with the formulation.

70. The method of claim 67, wherein one or more of the traits is decreased relative to a plant not treated with the formulation.

71. The method of claim 70, wherein the plant growth and/or the plant lifespan is

decreased relative to a plant not treated with the formulation.

72. The method of claim 70, wherein the fruit ripening time is decreased relative to a plant not treated with the formulation.

73. The method of claim 67, wherein the plant sensitivity to herbicide is increased relative to a plant not treated with the formulation.

74. A method of modulating a trait of an insect, comprising delivering an effective

amount of the formulation of claim 62 to the insect, to a plant infested with the insect, or to a lant prior to infestation with the insect.

75. The method of claim 74, wherein the trait modulated is insect growth, development, and/or lifespan.

76. A method of modulating the pathogenicity of a plant pathogen, comprising applying the formulation of claim 63 to the plant pathogen, to a plant infected with the plant pathogen, or to a plant prior to infection with the plant pathogen.

77. A plant cell, insect cell, fungal cell, bacterial cell, or mammalian cell comprising the modified polyethyleneimine of claim 1.

78. A method for screening capped polymer mixtures comprising:

(2) separately combining each of the at least two different mixtures of capped polymers with an oligo- or polynucleotide to form at least two different mixtures of oligo- or polynucleotide/capped polymer complexes; (3) separately assaying each of the at least two different mixtures of oligo- or polynucleotide/capped polymer complexes to determine the ability of each mixture to transfect a cell with the oligo- or polynucleotide.

79. The method of claim 78, further comprising:

(4) selecting an oligo- or polynucleotide/capped polymer complex mixture

determined to be able to transfect a cell with the oligo- or polynucleotide;

(5) separating the mixture into at least two oligo- or polynucleotide/capped

polymer complex sub-mixtures on the basis of average molecular weight;

(6) separately assaying each of the at least two different sub-mixtures of to

determine the ability of each sub-mixture to transfect a cell with the oligo- or polynucleotide.

80. The method of claim 78, wherein the polymer and the at least one cap precursor are reacted in three different ratios to form three different mixtures of capped polymers.

81. The method of claim 80, wherein the three different polymer/cap precursor ratios are 3:3, 3:2, and 3: 1.

82. The method of claim 78, wherein the oligo- or polynucleotide reduces the expression of a target gene expressed by the cell.

83. The method of claim 82, wherein the assaying comprises measuring expression levels of the target gene.

84. The method of claim 83, wherein the assaying comprises measuring mRNA levels of the target gene.

85. The method of claim 84, wherein the mRNA levels are measured by a method

selected from the group consisting of sequencing, quantitative PCR and Northern blot analysis.

86. The method of claim 83, wherein the assaying comprises measuring expression and/or activity of a protein encoded by the target gene.

87. The method of claim 86, wherein the expression of the protein is measured by a method selected from the group consisting of Western blot analysis, flow cytometry, ELISA and mass spectrometry.

88. The method of claim 86, wherein the fluorescence and/or enzyme activity of the protein is measured.

89. The method of claim 82, wherein the target gene is endogenous to the cell.

90. The method of claim 82, wherein the target gene is heterologous to the cell.

91. The method of claim 90, wherein the target gene encodes a marker protein.

92. The method of claim 91, wherein the marker protein is Firefly luciferase.

93. The method of claim 91, wherein the marker protein is Green Fluorescent Protein (GFP).

94. The method of claim 92, wherein the cell also expresses Renilla luciferase.

95. The method of claim 78, wherein the assaying comprises determining a phenotype of the cell, or an organism comprising the cell.

96. The method of claim 95, wherein the phenotype comprises changes in growth of the organism.

97. The method of any one of claims 78 to 96, wherein the oligo- or polynucleotide is an siRNA or an antisense RNA.

98. The method of claim 78, wherein the polymer comprising at least one primary and/or secondary amino group is a polyethyleneimine prepared by the ring-opening polymerization of aziridine, a polyethyleneimine derived from the partial or complete hydrolysis of a poly(2-alkyl-2-oxazoline), or combinations thereof.

99. The method of claim 78, wherein the poly(2-alkyl-2-oxazoline) is poly(2-ethyl-2- oxazoline).

100. The method of claim 78, wherein the at least one cap precursor is a compound of (Ilia):

wherein

R₅ is hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted hetero aliphatic or

heterocycloaliphatic group, an optionally substituted aralkyl

group, or an optionally substituted aryl group; and

R₆ is an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted hetero aliphatic or heterocycloaliphatic

group, an optionally substituted aralkyl group, or an optionally

substituted aryl group; a compound of formula (IVa):

wherein

are, independently, hydrogen or an optionally substituted aliphatic

group;

R9 is an optionally substituted aliphatic group; and X is O or N; or any combination thereof.