WO2009009434A1 - Polymers and uses thereof - Google Patents

Abstract

The present invention relates generally to the fields of molecular biology and biomolecule delivery. More specifically, the invention concerns are compositions and methods that utilize sulfonamide-containing polymers for gene transfection and delivery of other biomolecules.

Description

POLYMERS AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 60/948,301, filed July 6, 2007, which is incorporated by reference herein in its entirety.

ACKNOWLEDGEMENTS

This invention was made with government support under NIH Award Number RPl NS035680 awarded by National Institutes of Health (NIH). The government has certain rights in the invention.

BACKGROUND

Although gene transfer is perceived as a therapeutic strategy for treating a vast range of human diseases, including genetic disorders, cancer, and neurodegeneration (Pack et al, Nature Rev. Drug Discovery 4:581-593, 2005; Parker et al , Expert Rev. MoI Med. 5:1-14, 2003; Verma and Somia, Nature 389:239-242, 1997; Vile et al, Gene Ther. 7:2-8, 2000), a serious bottleneck in the development of gene therapy is the difficulty of efficient gene delivery and expression in target cells. Although gene delivery can be achieved by physical methods such as in vivo electroporation, ultrasound exposure, particle bombardment, and jet injection (Fechheimer et al., Proc. Natl. Acad. Sci. 84:8463-8467, 1987; Klein et al, Nature 327:70-73, 1987; Liu et al, Gene Ther. 6:1258-1266, 1999; Wong and Neumann, Biochem. Biophys. Res. Commun. 107:584-587, 1982), there are two methods that have been more widely investigated for gene delivery, namely, viral and non-viral vector approaches. Viral vectors are attractive for gene therapy because of their high efficiency (Verma and Somia, Nature 389:239-242, 1997; Thomas et al, Nature Rev., Genetics 4:346-358, 2003). But this approach exhibits many drawbacks, including the induction of immunological responses, random insertion of viral sequences into the host chromosomes, limitations on DNA encapsulation size, and high production cost (Pack et al, Nature Rev. Drug Discovery 4:581-593, 2005; Luo and Saltzman, Nature B iotechnol 18:33-37, 2000). Synthetic transfer vectors have attracted much interest because they offer advantages in improved safety, greater flexibility in structural modification, and low cost of synthesis. The major barriers to effective synthetic vectors, such as synthetic cationic polymer vectors, are their toxicity and low efficiency (Pack et al, Nature Rev. Drug Discovery 4:581-593, 2005; Parker et al, Expert Rev. MoI. Med. 5:1-14, 2003).

Nucleic acid transfer technologies are central to modern molecular analysis of brain function and development and offer great promise as human disease therapeutics. Preferred current methods of in vivo gene manipulation involve germ line genetics in model organisms, viral infection and, particularly for development studies, direct tissue electroporation. These approaches are inappropriate for human therapies, and even experimental animal models have many limitations. This poverty of in vivo methods stands in striking contrast to the many simple and effective methods of chemical transgenesis available for in vitro studies. There appears no obvious physical barrier to reagent-based plasmid transfer in vivo, but available cell culture compounds were developed for in vitro work and have only secondarily been screened for the low toxicity, efficacy and reliability needed for whole animal studies. The compositions and methods disclosed herein will provide a means to address such issues.

A variety of poly(ester-amines) (Anderson et al , Angew. Chem. Int. Ed. 42:3153- 3158, 2003; Lynn and Langer, J. Am. Chem. Soc. 122:10761, 2000; Lynn et al, J. Am. Chem. Soc. 123:8155, 2001; Akinc et al., J. Am. Chem. Soc. 125:5316-5323, 2003; Lynn et al, Angew. Chem. Int. Ed. 40(9): 1707-1710, 2001; Anderson et al., Proc. Natl. Acad. ScL 101(45): 16028-16033, 2004; Little et al, Proc. Natl. Acad. Sci. 101(26):9534-9539, 2004; Little et al, J. Controlled Release 107:449-462, 2005; Akinc et al, Bioconjugate Chem. 14:979-988, 2003) and poly(amido-amines) (Danusso and Ferruti, Polymer 11:88-113, 1970; Ferruti et al, Polymer 26:1336, 1985; Ferruti and Barbucci, Adv. Polym. Sci. 58:55- 92, 1984; Ferruti et al, Biomaterials 15:1235-1241, 1994; Ferruti et al, Macromol Chem. Phys. 200:1644-1654, 1999; Ferruti et al, Biomaterials 15:1235-1241, 1994; Hill et al, Biochim. Biophys. Acta 1427:161-174, 1999) have been prepared and used in biomedical applications, including gene delivery. Semi-automated synthesis and screening of a large library of degradable poly(ester-amines) have been achieved by R. Langer et al. (Angew. Chem. Int. Ed. 42:3153-3158, 2003).

Disclosed herein are compositions and methods that utilize sulfonamide-containing polymers for gene transfection and delivery of other biomolecules. Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and, together with the description, serve to explain the principles of the invention. These are non-limiting examples.

Figure 1 is a schematic showing a scheme for the synthesis of certain sulfonamide containing polymers disclosed herein. Shown is polymerization of divinylsulfonamides with bis(secondary amines), bis(primary amines) and mono(primary amine) via Michael addition reaction.

Figure 2 shows the structure of divinylsulfonamide and amine monomers for the initial screening library synthesis.

Figure 3 is a MALDI MS spectra and its expansion of a representative polymer E9.

Figure 4 is a graph of acid-base titration curves of representative polymers E3 (triangles) and E9 (circles), along with a NaCl control (squares).

Figure 5 is a photograph of gels from a gel retardation assay showing the electrophoretic shifts for plasmid DNA (pDNA) in the presence of different amounts of polymers.

Figure 6 is a pair of AFM images of pDNA only and polyplex (pDNA/ E3, 1 :2).

Figure 7 is a graph comparing poly(beta-aminosulfonamide) (PBAS) polyplex transfection efficacies in vitro with three commercially available transfection compounds.

Figure 8 is a group of micrographs showing polyplex transfection efficacies studied in vivo. (A-D) Examples of chick embryo midbrain screens carried out with the commercially available dendrimer SuperFect (A), the efficient PBAS polymers E9 (B) and E3/3900 Da (C), and the teratogen B17 (D). Illustrated are top views of embryonic day 5 chick embryos injected on embryonic day 2 with plasmid DNA-polymer mixtures. The injections were made into the midbrain vesicle, with the injection pipette directed ventrally. The plasmid DNA encodes the enzyme alkaline phosphatase, allowing transgenic cells to be detected in a histochemical procedure as dark purple cells (arrows, arrowheads). In the embryo wholemounts shown from a dorsal view, the dorsal midbrain has been cut away to show the labeled cells in the ventral midbrain. The polymers E9 and E3 were very efficient transfectants (++/+++, many positive cells) and compare very favorably with SuperFect (+, a few labeled cells). Polymer B17/2100 Da is the only PBAS teratogen tested so far to show transgenesis in vivo (arrow). The grossly misshapen head ("head") is illustrated at ³A angle. (E) Demonstration that intravascular delivery of E3 can elicit widespread labeling of the embryonic day 3 chick. Arrows point to some of the labeled cells. (F) Selective labeling of the lens (arrow) and otic placode with C12 polyplexes. Shown is a side view of an embryonic day 5 head. (G) Preliminary results with the TREN polymer T+l described below establish that it is effective at in vivo gene delivery (+++). Orientation as in panels A-C. (H) Tangential section through rat cortex in which EGFP expression plasmid complexed to Langer reagent B 14 elicited limited transfection of cells. The two spots illustrated are 1.5 mm apart and are part of a grid of cortical deposits delivered in this animal. In the experiment, tissue section chartings were made of the green fluorescence and in situ hybridization was carried out for EGFP mRNA to produce a permanent reaction product (arrows). These two spots, which coincided with sites of fluorescence, represent the best transfections in the disclosed cortex experiments.

Figure 9 is a graph comparing certain sulfonamide containing polymer transfection efficacies in vivo (black) and in vitro (light grey) demonstrating an almost complete dissociation. This indicates that in vivo screening as described herein is needed for identifying in vivo transfectants. Illustrated are four polymers (C6, C9, ClO and G4) that elicited no in vivo transfection, three polymers (E6, E8, E24) that elicited some transgenesis, and one polymer (E3, synthesized to 3900 or 4500 Da) that gave excellent transgenesis. Shown in light grey are transfection potencies for these polymers in vitro, measured by luciferase levels in COS-7 cells. Positive controls are the Langer compound B 14 (Akinc, et ah, J. Am. Chem. Soc. 125:5316-5323, 2003) and the cell culture product TransIT (Mirus Bio). Interestingly, the Langer B 14 compound was identified by Akinc et al. as an outstanding in vitro transfectant, but in a direct comparison is not as effective in vitro as some PBAS compounds.

Figure 10 is a schematic of one of the disclosed sulfonamide containing polymers functionalized with folate.

Figure 11 is a schematic showing transfection polymers with sulfonamide- containing side chains.

Figure 12 is a synthetic scheme for certain sulfonamide containing polymers with TREN model.

Figure 13 shows monomers for synthesis of certain sulfonamide containing polymers containing TREN.

Figure 14 is a synthetic scheme of PEI with a TREN group.

Figure 15 is a graph comparing PBAS polyplex transfection results in vivo (grey) and in vitro (black). The results demonstrated an almost complete dissociation in efficacy across the compounds, indicating that in vivo screening, as disclosed herein, is needed for identifying in vivo transfectants. Polymers are arrayed left to right according to in vitro transfection efficacy as measured by luciferase assays on COS-7 cells. In vivo polymer efficacies are shown following the rank order on the 0-+++ scale. Positive in vitro controls are the cell culture products TransIT-LTl (Mirus Bio), OmniPORTER (MP Biomedicals) and jetPEI (Polyplus). Note that the disclosed PBAS polymers (E24, C 18, C6, and E21) were more effective in vitro than either the commercial controls or the Langer polymer B 14 (Akinc, et al., J. Am. Chem. Soc. 125:5316-5323, 2003; B14 results not illustrated).

Figure 16 is a schematic of certain analogs of PBAS containing TREN. DETAILED DESCRIPTION OF THE INVENTION

The present disclosure describes, at least in part, the discovery of certain sulfonamide containing polymers. Disclosed herein are examples of sulfonamide containing polymers and some of their uses.

All patents, patent applications and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

It is to be understood that this invention is not limited to specific synthetic methods, or to specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, to specific pharmaceutical carriers, or to particular pharmaceutical formulations or administration regimens, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Definitions and Nomenclature

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture.

Throughout the specification and claims the word "comprise" and other forms of the word, such as "comprising" and "comprises," means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps. As used in the description and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, reference to "an agent" includes mixtures of two or more such agents, reference to "the polymer" includes mixtures of two or more such polymers, and the like.

The word "or" as used herein means any one member of a particular list and also includes any combination of members of that list.

Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independent of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value," and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed, then "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout the application data are provided in a number of different formats and that these data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weight of a particular element or component in a composition or article, denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms "substitution" or "substituted with" include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

A "residue" of a chemical species, as used in the specification and concluding claims, refers to the section or moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the section or moiety is actually obtained from the chemical species.

"A¹," "A²," "A³," and "A⁴" are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

The term "alkyl" as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can also be substituted or unsubstituted. The alkyl group can be substituted with one or more groups including, but not limited to, substituted or unsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein. A "lower alkyl" group is an alkyl group containing from one to six carbon atoms (e.g., methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the like).

The term "cycloalkyl" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The term "heterocycloalkyl" is a type of cycloalkyl group as defined above, and is included within the meaning of the term "cycloalkyl," where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, substituted or unsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term "polyalkylene group" as used herein is a group having two or more CH₂ groups linked to one another. The polyalkylene group can be represented by the formula — (CH₂)_a — , where "a" is an integer of from 2 to 500.

The term "alkoxy" as used herein is an alkyl or cycloalkyl group bonded through an ether linkage; that is, an "alkoxy" group can be defined as — OA¹ where A¹ is alkyl or cycloalkyl as defined above. "Alkoxy" also includes polymers of alkoxy groups as just described; that is, an alkoxy can be a polyether such as — OA — OA or — OA¹- (OA²)_a— OA³, where "a" is an integer of from 1 to 200 and A¹, A², and A³ are alkyl and/or cycloalkyl groups.

The term "alkenyl" as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (A¹A^C=C(A³A⁴) are intended to include both the E and Z isomers. This may be presumed in structural formulae herein wherein an asymmetric alkene is present, or it may be explicitly indicated by the bond symbol C=C. The alkenyl group can be substituted with one or more groups including, but not limited to, substituted or unsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein. The term "cycloalkenyl" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term "heterocycloalkenyl" is a type of cycloalkenyl group as defined above, and is included within the meaning of the term "cycloalkenyl," where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, substituted or unsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term "alkynyl" as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more groups including, but not limited to, substituted or unsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

The term "cycloalkynyl" as used herein is a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bound. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term "heterocycloalkynyl" is a type of cycloalkenyl group as defined above, and is included within the meaning of the term "cycloalkynyl," where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and heterocycloalkynyl group can be substituted or unsubstituted. The cycloalkynyl group and heterocycloalkynyl group can be substituted with one or more groups including, but not limited to, substituted or unsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term "aryl" as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term "aryl" also includes "heteroaryl," which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term "non-heteroaryl," which is also included in the term "aryl," defines a group that contains an aromatic group that does not contain a heteroatom. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, substituted or unsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term "biaryl" is a specific type of aryl group and is included in the definition of "aryl." Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

The term "aldehyde" as used herein is represented by the formula — C(O)H. Throughout this specification "C(O)" is a short hand notation for a carbonyl group, i.e., C=O.

The terms "amine" or "amino" as used herein are represented by the formula NA¹A²A³, where A¹, A², and A³ can be, independently, hydrogen or substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term "polyamine" is included within the meaning of "amine" and has the formula — (A¹ — NA² — A³)_a — , where where A¹ and A³ can be, independently, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein, A² can be a hydrogen, substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein, and "a" is an integer from 1 to 10,000.

The term "carboxylic acid" as used herein is represented by the formula — C(O)OH. The term "carboxylate" is a carboxylic acid that has been deprotonated, i.e., — C(O)O^". Protonation and deprotonation can be achieved by changes in pH. The terms "carboxylic acid" and "carboxylate" are understood to be interchangeable.

The term "carbonate" as used herein is represented by the formula — OC(O)O — . A "polycarbonate" as used herein is included within the meaning of "carbonate" and is represented by the formula — (A¹0C(0)0-A²)_a — , where A¹ and A² can be, independently, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and "a" is an integer from 1 to 10,000.

The term "ester" as used herein is represented by the formula — OC(O)A or — C(O)OA¹, where A¹ can be a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term "polyester" as used herein is represented by the formula — (A'θ(O)C-A²-C(O)O)_a — or — (A¹O(O)C-A²-OC(O))_a — , where A¹ and A² can be, independently, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and "a" is an integer from 1 to 10,000. "Polyester" is as the term used to describe a group that is produced by the reaction between a compound having at least two carboxylic acid groups with a compound having at least two hydroxyl groups.

The term "ether" as used herein is represented by the formula A OA , where A and A² can be, independently, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein. The term "polyether" as used herein is represented by the formula — (A¹O-A²O)₈ — , where A¹ and A² can be, independently, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and "a" is an integer of from 1 to 10,000. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.

The term "halide" as used herein refers to the halogens fluorine, chlorine, bromine, and iodine.

The term "hydroxyl" as used herein is represented by the formula — OH.

The term "ketone" as used herein is represented by the formula A¹C(O)A², where A¹ and A² can be, independently, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

The term "azide" as used herein is represented by the formula — N₃.

The term "nitro" as used herein is represented by the formula — NO₂.

The term "nitrile" as used herein is represented by the formula — CN.

The term "silyl" as used herein is represented by the formula — SiA A A³, where A¹, A², and A³ can be, independently, hydrogen or a substituted or unsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term "sulfo-oxo" as used herein is represented by the formulas — S(O)A¹, -S(O)₂A¹, -OS(O)₂A¹, Or-OS(O)₂OA¹, where A¹ can be hydrogen or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. Throughout this specification "S(O)" is a short hand notation for S=O. The term "sulfonyl" is used herein to refer to the sulfo-oxo group represented by the formula -S(O)₂A¹, where A¹ can be hydrogen or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term "sulfone" as used herein is represented by the formula A¹S(O)₂A², where A¹ and A² can be, independently, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term "sulfoxide" as used herein is represented by the formula A¹S(O)A², where A¹ and A² can be, independently, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

The term "sulfonamide" as used herein is represented by the formula — S(O)₂NA¹ — , where A¹ can be hydrogen, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

The term "thiol" as used herein is represented by the formula — SH.

Many suitable multi-armed polymers are referred to as dendrimers. The term "dendrimer" means a branched polymer that possesses multiple generations, where each generation creates multiple branch points. "Dendrimers" can include dendrimers having defects in the branching structure, dendrimers having an incomplete degree of branching, crosslinked and uncrosslinked dendrimers, asymmetrically branched dendrimers, star polymers, highly branched polymers, highly branched copolymers and/or block copolymers of highly branched and not highly branched polymers.

"R¹," "R²," "R³," "Rⁿ," "L," "Q," "W," "X," "Yⁿ," and "Zⁿ" as used herein can, independently, possess one or more of the groups listed above. For example, if R¹' is a polyether group, one of the hydrogen atoms of the polyether group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or, alternatively, the first group can be pendant (i.e., attached) to the second group. For example, with the phrase "a polyether group comprising an alkene group," the alkene group can be incorporated within the backbone of the polyether group. Alternatively, the alkene group can be attached to the backbone of the polyether group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.

"Biomolecule" as used herein refers to a portion of the disclosed compositions. For example, a "biomolecule" can be associated with a sulfonamide-containing polymer to form the disclosed compositions. A "biomolecule" can be a naturally-occuring or synthetic substance. A "biomolecule" can be a polypeptide, peptidomimetic, nucleic acid, or a small molecule. For example, a biomolecule can be a protein, an oligosaccharide, a polysaccharide, DNA, RNA, siRNA, mRNA, microRNA, an antibody, an antibody fragment, a sugar, a lipid, a vitamin, or a chemotherapeutic agent.

A "chemotherapeutic agent" is a chemical compound useful in the treatment of a disease, such as cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN(R) (cyclosphosphamide); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9- tetrahydrocannabinol (dronabinol, MARINOL(R)); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN(R)), CPT-Il (irinotecan, CAMPTOSAR(R)), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBl-TMl); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosf amide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Intl. Ed. Engl, 33:183-186, 1994); dynemicin, including dynemicin A, an esperamicin, as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN(R), morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino- doxorubicin, doxorubicin HCl liposome injection (DOXIL(R) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR(R)), tegafur (UFTORAL(R)), capecitabine (XELODA(R)), an epothilone, and 5-fluorouracil (5- FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK(R) polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISEME(R), FILDESIN(R)); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, e.g., paclitaxel (TAXOL(R)), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE(TM)), and doxetaxel (TAXOTERE(R)); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELB AN(R)); platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine (ONCOVIN(R)); oxaliplatin; leucovovin; vinorelbine (NAVELBINE(R)); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATESf(TM)) combined with 5-FU and leucovovin.

"Polypeptide" as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules.

In addition, as used herein, the term "polypeptide" refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative,.covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Proteins - Structure and Molecular Properties 2^nd Ed., T.E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).

As used herein, the term "amino acid sequence" refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D, aspartic acid (aspartate); E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid; and X, any amino acid. As used herein, "peptidomimetic" means a mimetic of a peptide which includes some alteration of the normal peptide chemistry. Peptidomimetics typically enhance some property of the original peptide, such as increased stability, increased efficacy, enhanced delivery, increased half life, etc. Methods of making peptidomimetics based upon a known polypeptide sequence is described, for example, in U.S. Patent Nos. 5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position. One embodiment of the present invention is a peptidomimetic wherein the compound has a bond, a peptide backbone or an amino acid component replaced with a suitable mimic. Some non-limiting examples of unnatural amino acids which may be suitable amino acid mimics include /3-alanine, L-ct- amino butyric acid, L-γ-amino butyric acid, L-α-amino isobutyric acid, L-e -amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic acid, N-e-Boc-N-α-CBZ-L-lysine, N-e-Boc-N-α-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-α-Boc- N-δCBZ-L-ornithine, N-δ-Boc-N-α-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc- hydroxyproline, and Boc-L-thioproline.

The phrase "nucleic acid" as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single- stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, microRNA, shRNA, or siRNA, or any combination thereof.

By "sample" is meant an animal; a tissue or organ from an animal; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g. a polypeptide or nucleic acid). A sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells or cell components.

By "modulate" is meant to alter, by increasing or decreasing.

By "subject" is meant an individual. Thus, the "subject" can include domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), fish (e.g. farmed salmon), invertibrates (e.g. snails) and birds. In one aspect, the subject is a mammal such as a primate or a human. A "subject" can also be a plant or other agricultural product. For example, the disclosed compositions can be used to transform plants, thereby providing transformed plants.

By an "effective amount" of a compound as provided herein is meant a sufficient amount of the compound to provide the desired effect. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of disease (or underlying genetic defect) that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact "effective amount." However, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

By "isolated polypeptide" or "purified polypeptide" is meant a polypeptide (or a fragment thereof) that is substantially free from the materials with which the polypeptide is normally associated in nature. The polypeptides of the invention, or fragments thereof, can be obtained, for example, by extraction from a natural source (for example, a mammalian cell), by expression of a recombinant nucleic acid encoding the polypeptide (for example, in a cell or in a cell-free translation system), or by chemically synthesizing the polypeptide. In addition, polypeptide fragments may be obtained by any of these methods, or by cleaving full length polypeptides.

By "isolated nucleic acid" or "purified nucleic acid" is meant DNA that is free of the genes that, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, such as an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryote or eukaryote (e.g., a transgene); or which exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR, restriction endonuclease digestion, or chemical or in vitro synthesis). It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. The term "isolated nucleic acid" also refers to RNA, e.g., an RNA molecule that is encoded by an isolated DNA molecule, or that is chemically synthesized, or that is separated or substantially free from at least some cellular components, for example, other types of RNA molecules or polypeptide molecules.

By a "transgene" is meant a nucleic acid sequence that is inserted by artifice into a cell and either becomes a part of the genome of that cell or is maintained for a period of time as an episome. Such transgenes may be stably maintained by the cell, and thus passed on to progeny cells, or transiently maintained. Such a transgene may be (but is not necessarily) partly or entirely heterologous (for example, derived from a different species) to the cell.

By "transgenic animal" is meant an animal comprising a transgene as described above. Transgenic animals are made by techniques that are well known in the art.

By "transformed plant" or "transgenic plant" is meant a plant or agricultural product comprising a transgene as described above. Transformed plants are made by techniques that are well known in the art.

By "knockout mutation" is meant an alteration in the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% relative to the unmutated gene. The mutation may, without limitation, be an insertion, deletion, frameshift, or missense mutation. A "knockout animal," for example, a knockout mouse, is an animal containing a knockout mutation. The knockout animal may be heterozygous or homozygous for the knockout mutation. Such knockout animals are generated by techniques that are well known in the art.

By "knockdown mutation" is meant an alteration in the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 10% relative and at most 80% relative to the unmutated gene. The mutation may, without limitation, be an insertion, deletion, frameshift, or missense mutation.

By "treat" is meant to administer a compound or molecule of the invention to a subject, such as a human or other mammal (for example, an animal model), that has an increased susceptibility for developing a disease or condition, or that has a disease or condition, in order to prevent or delay a worsening of the effects of the disease or condition, or to partially or fully reverse the effects of the disease or condition.

By "prevent" is meant to minimize the chance that a subject who has an increased susceptibility for developing a particular condition will develop that condition.

"Specifically binds" encompasses the biological interaction of a compound or molecule with a target molecule. Examples of such biological interactions includes, but is not limited to, antibody-antigen interactions, receptor-ligand interactions, and any other interaction between biologically relevant binding partners.

By "probe," "primer," or "oligonucleotide" is meant a single-stranded DNA or RNA molecule of defined sequence that can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the "target"). The stability of the resulting hybrid depends upon the extent of the base-pairing that occurs. The extent of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecules and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art. Probes or primers specific for nucleic acids (for example, genes and/or mRNAs) have at least 80%-90% sequence complementarity, preferably at least 91%-95% sequence complementarity, more preferably at least 96%-99% sequence complementarity, and most preferably 100% sequence complementarity to the region of the nucleic acid to which they hybridize. Probes, primers, and oligonucleotides may be detectably-labeled, either radioactively, or non-radioactively, by methods well- known to those skilled in the art. Probes, primers, and oligonucleotides are used for methods involving nucleic acid hybridization, such as: nucleic acid sequencing, reverse transcription and/or nucleic acid amplification by the polymerase chain reaction, single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMSA).

By "specifically hybridizes" is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (that is, base-pairs) with a substantially complementary nucleic acid under high stringency conditions, and does not substantially base pair with other nucleic acids.

By "high stringency conditions" is meant conditions that allow hybridization comparable with that resulting from the use of a DNA probe of at least 40 nucleotides in length, in a buffer containing 0.5 M NaHPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at a temperature of 65⁰C, or a buffer containing 48% formamide, 4.8X SSC, 0.2 M Tris-Cl, pH 7.6, IX Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42⁰C. Other conditions for high stringency hybridization, such as for PCR, northern, Southern, or in situ hybridization, DNA sequencing, etc., are well-known by those skilled in the art of molecular biology. (See, for example, F. Ausubel et ah, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998).

The term "molecule of interest" as used herein refers to a biological molecule that is either naturally present in a cell or subject or has been previously introduced into a cell or subject. A "molecule of interest" can be a polypeptide, peptidomimetic, nucleic acid, or a small molecule. For example, a molecule of interest can be a protein, an oligosaccharide, a polysaccharide, DNA, RNA, siRNA, mRNA, microRNA, or a vitamin. A "molecule of interest" is not meant to refer to a portion of the disclosed compositions, however, once the biomolecule of the disclosed compositions is introduced into a cell or subject and is no longer part of the disclosed compositions, it can then be deemed as a "molecule of interest".

The term "molecule of interest" or "polynucleotide of interest" can mean a nucleic acid sequence (e.g., a therapeutic gene), that is partly or entirely heterologous, i.e., foreign, to a cell or subject into which it is introduced.

The term "molecule of interest" or "polynucleotide of interest" can also mean a nucleic acid sequence, that is partly or entirely homologous to an endogenous gene of the cell or subject into which it is introduced, but which is designed to be inserted into the genome of the cell or subject in such a way as to alter the genome (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in "a knockout mutation" or "a knockdown mutation"). For example, a "molecule of interest" or a "polynucleotide of interest" can be cDNA, DNA, or mRNA.

The term "molecule of interest" or "polynucleotide of interest" can also mean a nucleic acid sequence, that is partly or entirely complementary to an endogenous gene of the cell or subject into which it is introduced. For example, the "molecule of interest" or "polynucleotide of interest" can be microRNA, shRNA, or siRNA.

A "molecule of interest" or "polypeptide of interest" can mean a polypeptide sequence (e.g., a therapeutic protein), that is partly or entirely heterologous, i.e., foreign, to a cell or subject into which it is introduced.

A "molecule of interest" or "polypeptide of interest" can also mean a polypeptide sequence (e.g., a therapeutic protein), that is expressed from a "gene of interest".

A "molecule of interest" or "polypeptide of interest" can also mean a polypeptide sequence, that is partly or entirely homologous to an endogenous polypeptide of the cell or subject into which it is introduced, but which is designed to be inserted into the cell or subject in such a way as to alter or modulate the endogenous levels or activity of the "polypeptide of interest". For example, a "molecule of interest" or a "polypeptide of interest" can be a peptidomimetic.

Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures. Compositions

Disclosed herein are materials, compounds, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a composition is disclosed and a number of modifications that can be made to a number of components of the composition are discussed, each and every combination and permutation that is possible is contemplated unless specifically indicated to the contrary. Thus, if a class of components or moieties A, B, and C are disclosed as well as a class of components or moieties D, E, and F and an example of a composition A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific aspect or combination of aspects of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

As described above, the disclosed polymers can be used to deliver a variety of molecular entities such as nucleic acids, peptides, small molecules, etc. The disclosed compositions can be used for, among other things, in vivo gene manipulation. These compositions comprise one or more biomolecules with a sulfonamide-containing polymer. The sulfonamide-containing polymers are basic and can be protonated in certain pH values, which help their interaction with biomolecules like DNA. Also, the disclosed sulfonamide- containing polymers are biodegradable. The disclosed compositions can contain one or more different kinds of sulfonamide-containing polymers as disclosed herien. In the disclosed compositions and methods, the sulfonamide-containing polymers can be characterized as polymers comprising Formula I:

— [— [multi-sulf onamide] — [linker]—] — n Formula I where the multi-sulfonamide component is a residue of a multi-sulfonamide monomer that contains two or more sulfonamide moieties, and the linker component is a residue of a linker monomer that links one multi-sulfonamide residue to another. The variable "n" can be any integer, e.g., from 2 to 10,000.

The molecular weight of the sulfonamide-containing polymer can vary and will depend upon the selection of the monomers and the particular application. In one example, the sulfonamide-containing polymer can have a molecular weight of from about 500 Da to about 1,000,000 Da. In another aspect, the molecular weight of the polymer can be about 1,000; 2,500; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 75,000; 100,000; 200,000; 250,000; 300,000; 350,000; 400,000; 450,000; 500,000; 550,000; 600,000; 650,000; 700,000; 750,000; 800,000; 850,000; 900,000; 950,000; or 1,000,000 Da, where any stated values can form a lower and/or upper endpoint of a molecular weight range as appropriate. In a specific example, the molecular weight of the disclosed sulfonamide-containing polymers is from about 1,000 Da to about 8,500 Da. In other examples, the disclosed sulfonamide containing polymers have a molecular weight of less than 5,000 Da. The size of the disclosed compositions, which includes the one or more biomolecule and/or bioactive molecule and the sulfonamide-containing polymer is generally on the nanometer scale, for example from about 1 to about 1000 run, from about 10 to about 900 nm, from about 50 to about 800 nm, from about 100 to about 700 nm, from about 150 to about 600 nm, from about 200 to about 500 nm, from about 500 to about 1000 nm, or from about 150 to about 200 nm in diameter.

The monomers used to prepare the disclosed sulfonamide-containing polymers are flexible in that they can be readily modified. And the polymerization process utilizes simple reactions that can, in most instances, be adopted for combinatorial synthesis to generate a large library of polymers for screening. Multi-sulfonamide monomers and residues thereof

In many examples, the multi-sulfonamide monomer and residue thereof can contain two or more sulfonamide moieties. When the multi-sulfonamide monomer and residue thereof contains two sulfonamide moieties it is termed a "disulfonamide." It is contemplated, however, that the multi-sulfonamide residue can contain three, four, five, six, seven, eight, nine, ten, or more sulfonamide residues. Also, as is described elsewhere herein, the disclosed sulfonamide-containing polymers can be prepared by polymerizing a multi-sulfonamide monomer with a linker monomer. As such, the multi-sulfonamide monomers disclosed herein (and residues therefrom) have, in addition to two or more sulfonamide moieties, one or more moieties that are capable of forming a bond with a linker monomer.

In the disclosed sulfonamide-containing polymers, a multi-sulfonamide residue is said to be "derived from" a multi-sulfonamide monomer. By "derived from" it is meant that a residue is the portion of a monomer that remains after the monomer reacts in a polymerization reaction.

Examples of suitable multi-sulfonamide monomers that can be used to form a sulfonamide-containing polymer are represented by Formula II:

Formula II where Z¹ and Z² are the same or different and are functional groups capable of forming a bond with a linker monomer; where R and R are, independent of one another, hydrogen, substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol; and X can comprise a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol.

Z functional groups

Coupling a multi-sulfonamide monomer to a linker monomer in a polymerization reaction can be accomplished by any reaction that can form a bond between the multi- sulfonamide monomer and the linker monomer. In Formula II, the substituents Z¹ and Z² (referred to together as "Z substituents" herein) represent functional groups or moieties on the multi-sulfonamide monomer that can form a bond with the linker monomer. As such, the Z substituents are generally reactive. Each multi-sulfonamide monomer can have the same or different Z-substituents, i.e., there can be more than one type of moiety capable of forming a bond with a linker monomer. Also, though not shown in Formula II, the substituent X can contain one or more functional groups or moieties capable of forming a bond with a linker monomer. Further, more than one type of multi-sulfonamide monomer, each differing in their respective Z substituents, can be used to form the disclosed sulfonamide-containing polymers. That is, the sulfonamide-containing polymers disclosed herein can contain more than one different types of multi-sulfonamide residues.

In some examples, a Z substituent can have one or more electrophilic functional groups that can react with one or more nucleophilic functional groups on a linker monomer to form a bond. Alternatively, a Z substituent can have one or more nucleophilic functional groups that can react with one or more electrophilic functional groups on a linker monomer to form a bond. By "nucleophilic functional group" is meant any moiety that contains or can be made to contain an electron rich atom; examples of nucleophilic functional groups are disclosed herein. By "electrophilic functional group" is meant any moiety that contains or can be made to contain an electron deficient atom; examples of electrophilic functional groups are also disclosed herein.

Nucleophilic functional groups on the multi-sulfonamide monomer

In a particular example, a Z substituent on the multi-sulfonamide monomers of Formula II can be a moiety that comprises one or more nucleophilic functional groups, which can react with an electrophilic group on a linker monomer to form a bond between the resulting multi-sulfonamide residue and linker residue. It is understood that when a nucleophilic functional group is reacted with an electrophilic functional group, the nucleophilic functional group is no longer nucleophilic or at least less nucleophilic than before the reaction and a bond between the nucleophilic and electrophilic functional groups results. In this sense, the disclosed sulfonamide-containing polymers can, in some examples, be without a nucleophilic functional group on the multi-sulfonamide residue; that is, the nucleophilic functional group has been coupled to an electrophilic functional group on the linker monomer and is no longer nucleophilic or as nucleophilic as before. However, for the purposes of this disclosure, various functional groups are identified by referring to them prior to bond formation. Examples of nucleophilic functional groups that can be present on a multi-sulfonamide monomer {i.e., a Z substituent) include, but are not limited to, an amine, amide, hydroxyl, or thiol group, as are described herein.

In Formula II, a Z substituent {e.g., either Z¹ or Z² or both) can be an amine. In this instance, the amine acts as a nucleophilic functional group that can react with an electrophilic moiety on a linker monomer {e.g., reacting with an alkenyl or alkynyl adjacent to an electron withdrawing group, halide, aldehyde, ketone, ester, acid anhydride, acid halide, or isocyanate to form an amine, imine, amide, or urea bond). For example, either Z¹ or Z² or both can be a primary amine {i.e., — NH₂), a secondary amine {i.e., — NHR¹⁰), or a tertiary amine ( — NR¹⁰ ₂), where each R¹⁰ is, independent of the others, a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol. In some specific examples, a Z substituent can be a cyclic amine, such as aziridinyl, azetidinyl, pyrrolidinyl, pyrrolinyl, pyrazolidinyl, imidazolidinyl, pyrazolinyl, piperidinyl, piperazinyl, indolinyl, or morpholinyl group.

In one specific example, either Z¹ or Z² or both on the multi-sulfonamide monomers can comprise a polymeric amine (i.e., a polymer that comprises one or more amine groups). Suitable examples of polymeric amines are amino-acid-based polymers. As used herein "amino acid" means the typically encountered twenty amino acids that make up polypeptides. In addition, it further includes less typical constituents that are both naturally occurring, such as, but not limited to, formylmethionine and selenocysteine, analogs of typically found amino acids, and mimetics of amino acids or amino acid functionalities. Some specific examples of amino-acid-based polymers that are suitable include, but are not limited to, polylysine, proteins (e.g., enzymes), and peptides, including mixtures thereof.

Other suitable examples of polymeric amines are olefin based polymers that contain one or more amine functional group. Many such polyamines can be obtained commercially or can be prepared by methods known in the art. Suitable examples of polyamines that can be present on a multi-sulfonamide monomer (i.e., a Z substituent) include, but are not limited to, polyvinyl amine and polyalkyleneimines like polyethyleneimine.

Still further examples of polymeric amines are polyamides that are prepared by the condensation of a diamine monomer with a diacid or diester monomer. Such polyamides are well known in the art and can be obtained commercially. Alternatively, polyamides can be prepared by self condensation of a monomer containing an amine and an acid or ester functional group, or through a ring opening reaction of a cyclic amide (i.e., lactam) such as caprolactam.

Yet another example of a suitable polymeric amine is a polyether amine, which is understood to be included within the meaning of a polymeric amine. Polyether amines contain primary amino groups attached to the terminus of a polyether backbone. The polyether backbone is typically based either on propylene oxide (PO), ethylene oxide (EO), or mixed EO/PO. In one example, the polyether amine can be a polyoxyalkyleneamines. Such polyether amines can be obtained commercially.

A still further example of a suitable polymeric amine is a dendrimeric polyamine. Such dendrimers, in one example, have a macromolecular architecture called "dense star" polymers. Unlike classical polymers, these dendrimers have a high degree of molecular uniformity, narrow molecular weight distribution, specific size, and shape characteristics, and a highly-functionalized terminal surface. An example of dendrimeric polyamines are the PAMAM dendrimers, which are poly(amidoamine) dendrimers. The manufacturing process for these dendrimers is a series of repetitive steps starting with a central initiator core (e.g., ethylenediamine core). Each subsequent growth step represents a new "generation" of polymer with a larger molecular diameter, twice the number of reactive surface sites, and approximately double the molecular weight of the preceding generation.

In other similar examples, either Z¹ or Z² or both can be an amide (i.e., — NR¹¹C(O)R¹¹), where each R¹¹ is, independent of the others, hydrogen, or is a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol.

In yet another suitable example of a nucleophilic Z substituent in the multi- sulfonamide monomers of Formula II, either Z¹ or Z² or both can be an alcohol. In this instance, the alcohol acts as a nucleophilic functional group that can react with an electrophilic moiety on a linker monomer (e.g. , reacting with an alkenyl or alkynyl adjacent to an electron withdrawing group, halide, aldehyde, ketone, ester, acid anhydride, acid halide, or isocyanate to form an ether, ester, acetal, carbonate, or urethane bond). For example, either Z¹ or Z² or both can be a primary alcohol, a secondary alcohol, or a tertiary alcohol, i.e.,

where each R¹², alone or in combination with another R¹² group, is a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol, and wherein one of the R¹² substituents is connected to the multi-sulfonamide monomer.

In yet another suitable example of a nucleophilic Z substituent in the multi- sulfonamide monomers of Formula II, either Z¹ or Z² or both can be a polymeric alcohol (i.e., a polymer that comprises one or more hydroxyl groups). Such hydroxyl groups can react with electrophilic groups on a linker monomer to form a bond. A suitable polymeric alcohol is polyvinyl alcohol, which is commercially available or can be prepared by the hydrolysis of polyvinyl acetate. Other suitable polymeric alcohols include, but are not limited to, l,l,l-tris(hydroxymethyl)propanyl (TMP), diglycerol, hyperbranched polyglycerol, and the like.

Also contemplated are multi-sulfonamide monomers where either Z¹ or Z² or both are thiols ( — SH), including primary, secondary, tertiary, dendrimeric, and polymeric thiols that correspond to the alcohols disclosed herein.

Electrophilic functional groups on the multi-sulfonamide monomer

In other examples of multi-sulfonamide monomers disclosed herein, a Z substituent (e.g., either Z¹ or Z² or both) can be a moiety that comprises one or more electrophilic functional groups, which can react with a nucleophilic group on a linker monomer to form a bond between the resulting multi-sulfonamide residue and linker residue. It is understood that when an electrophilic functional group is reacted with a nucleophilic functional group, the electrophilic functional group is no longer electrophilic or is at least less electrophilic than before the reaction and a bond between the electrophilic and nucleophilic functional groups results. In this sense, the disclosed sulfonamide-containing polymers can, in some examples, be without an electrophilic functional group on the multi-sulfonamide residue; that is, the electrophilic functional group has been coupled to a nucleophilic functional group on the linker monomer and is no longer electrophilic or as electrophilic as before. Examples of suitable electrophilic functional groups that can be used include, but are not limited to, an alkenyl or alkynyl moiety adjacent to an electron withdrawing group, halide, aldehyde, ketone, ester, acid anhydride, acid halide, or isocyanate groups. In one specific example, the suitable electrophilic functional group for a Z substituent in Formula II, is a alkenyl or alkynyl moiety adjacent to a sulfonamide moiety.

In another example, a suitable electrophilic Z substituent can be an ester or an acid. In yet another example, a suitable electrophilic Z substituent can be a polyester {i.e., a polymer that comprises one or more ester groups). Polyesters are well known and can be obtained commercially or by methods known in the art. Suitable examples of polyesters include, but are not limited to, polyalkylene terephthalates, and polycycloalkylene alkyl esters.

Multi-sulfonamide monomers where the Z substituent comprises succinimidyl ester moieties can also be used to react with linker monomers that contain amine, carboxylate, alcohol, or thiol functional groups. Succinimidyl ester containing multi-sulfonamide monomers are particularly reactive towards amines, where the resulting amide bond that is formed is as stable as a peptide bond. However, some succinimidyl ester containing multi- sulfonamide monomers may not be compatible with a specific application because they can be quite insoluble in aqueous solution. To overcome this limitation, sulfosuccinimidyl ester containing multi-sulfonamide monomers, which typically have higher water solubility than succinimidyl ester containing multi-sulfonamide monomers, can be used.

Sulfosuccinimidyl ester containing multi-sulfonamide monomers can generally be prepared in situ from simple carboxylic acid containing multi-sulfonamide monomer by dissolving the multi-sulfonamide monomer in an amine-free buffer that contains N- hydroxysulfosuccinimide and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Also, 4- sulfo-2,3,5,6-tetrafluorophenol (STP) ester containing multi-sulfonamide monomer can be prepared from 4-sulfo-2,3,5,6-tetrafluorophenol in the same way as sulfosuccinimidyl ester containing linker monomers.

In still other examples, one or more of the electrophilic Z substituents can comprise a halide. One or more of the Z substituents can also comprise an alkyl, cycloalkyl, cycloheteroalkyl, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, or heteroaryl moiety that contains a halide. Such moieties can react with linker monomers that contain amine, carboxylate, alcohol, or thiol functional groups to form, for example, amine, ester, ether, or sulfide bonds, respectively.

In still further examples, one or more of the electrophilic Z substituents can comprise an aldehyde, a ketone, an acid anhydride, or an acid halide moiety. Z substituents that are aldehydes can react with linker monomers that contain amines to form Schiff bases.

In yet another example, one or more of the electrophilic Z substituents can comprise an isocyanate moiety. Isocyanates are readily derivable from acyl azides, and they react with amine containing linker monomers to form ureas, they react with alcohol containing linker monomers to form urethanes, and they react with thiol containing linker monomers to form thiourethanes.

Isothiocyanate moieties are an alternative to isocyanates and are moderately reactive but quite stable in water. Multi-sulfonamide monomers containing isothiocyanates will react with an amine, alcohol, or thiol containing linker monomer to form thioureas and thiourethanes.

Also, when a Z substituent is not generally reactive it can be converted into a more reactive moiety. For example, Z substituents that comprise carboxylate or carboxylic acid groups can, depending on the conditions, be slow to react with a nucleophilic substituent on a linker monomer. However, these Z substituents can be converted into more reactive, activated esters by a carbodiimide coupling with a suitable alcohol, e.g., 4-sulfo-2,3,5,6- tetrafluorophenol, N-hydroxysuccinimide or N-hydroxysulfosuccinimide. This results in a more reactive, water-soluble activated ester moiety. Various other activating reagents that can be used for the coupling reaction include, but are not limited to, l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), N,N' - diisopropyl-carbodiimide (DIP), benzotriazol- 1 -yl-oxy-tris-(dimethylamino)phosphonium hexa-fluorophosphate (BOP), hydroxybenzotriazole (HOBt), and N-methylmorpholine (NMM), including mixtures thereof.

X moiety

In some examples, the moiety X in Formula II can comprise a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol. In one example, the X moiety is substituted with a further Z substituent that is capable of forming a bond with a linker monomer.

, The X moiety can, in further specific examples, comprise a substituted or unsubstituted C₁-C₆ branched or straight-chain alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, sec-pentyl, or hexyl. In a specific example, the X moiety can comprise a polyalkylene (i.e., -(CH₂)_n-, wherein n is from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 1 to 5, from 1 to 3, or from 10 to 20). Still further, the moiety X in Formula II can comprise a cycloalkyl, such as cyclopentyl, cyclohexyl, or cyclopropyl. The moiety X can also be a cycloheteroalkyl like piperazine, 2-methylpiperazine, l,3-di(piperidin-4-yl)propane, pyranyl, and the like.

In another example, the moiety X in Formula II can comprise a C₁-C₆ branched or straight-chain alkoxy such as a methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, neopentoxy, sec-pentoxy, or hexoxy. In still other examples, the moiety X in Formula II can comprise a C₂-C₆ branched or straight-chain alkyl, wherein one or more of the carbon atoms are substituted with oxygen (e.g., an ether), sulfur (e.g., a thioether), or nitrogen (e.g., an amino). For example, a suitable moiety X can include, but is not limited to, a methoxymethyl, methoxyethyl, methoxypropyl, methoxybutyl, ethoxymethyl, ethoxyethyl, ethoxypropyl, propoxymethyl, propoxyethyl, methylarninomethyl, methylaminoethyl, methylaminopropyl, methylaminobutyl, ethylaminomethyl, ethylaminoethyl, ethylaminopropyl, propylaminomethyl, propylaminoethyl, methoxymethoxymethyl, ethoxymethoxymethyl, methoxyethoxymethyl, methoxymethoxyethyl, and the like, and derivatives thereof.

In one specific example, X can be -CH₂CH₂NR²¹CH₂CH₂-, or -CH₂CH₂CHR²¹CH₂CH₂-, where R²¹ is -CH₂CH₂NHSO₂-p-CH₃-Ph (Ph is phenyl).

Specific examples of ntulti-sulfonamide monomers

Suitable multi-sulfonamide monomers are readily commercially available and/or can be synthesized by those of ordinary skill in the art. And the particular multi-sulfonamide monomer that can be used to prepare the disclosed sulfonamide-containing polymers can be chosen by one of ordinary skill in the art based on factors such as cost, convenience, availability, compatibility with various reaction conditions, the type of linker monomer with which the multi-sulfonamide monomer is to interact, and the like.

In certain examples, the multi-sulfonamide monomer can have Formula II, where Z is an electrophilic moiety and X is a substituted or unsubstituted alkyl group. Specific examples of such compounds are shown below in Formula H-I.

where each R⁶, independent of the others, is hydrogen, or is a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol; and m is from 1 to 25.

Other examples of multi-sulfonamide monomers are those having Formula II, where Z is an electrophilic moiety and X is a substituted or unsubstituted cycloalkyl or cycloheteroalkyl group. Specific examples of these are shown below in Formulae II-II through H-X.

where R m arse as described before, and Q is NR⁶, O, S, or CH₂. In these monomers, cis and trans isomers are contemplated herein.

In a still further example, the multi-sulfonamide can have Formula II, wherein Z is an electrophilic moiety and X is an ether or polyether, for example a compound having Formula II-XI below.

where R⁶ and m are as described before.

In certain specific examples, the multi-sulfonamide monomer can be any of the monomers A through G shown below.

In several particular examples, the X moiety can be a derivative of Tris(2- aminoethyl)amine (TREN). Thus, a suitable multi-sulfonamide monomer can be the monomer T:

Linker monomers and residues thereof

As noted herein, the disclosed sulfonamide-containing polymers can be prepared by polymerizing a multi-sulfonamide monomer with a linker monomer. As such, the linker monomers disclosed herein (and residues therefrom) have one or more moieties capable of forming a bond with a multi-sulfonamide monomer.

In the disclosed sulfonamide-containing polymers, a linker residue is said to be "derived from" a linker monomer. By "derived from" is meant that a residue is the portion of a monomer that remains after the monomer reacts in a polymerization reaction. Examples of suitable linker monomers that can be used to form a sulfonamide-containing polymer are represented by

Y¹— L— Y² Formula III where Y¹ and Y² are the same or different and are functional groups capable of forming a bond with a multi-sulfonamide monomer and where L can comprise a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol.

The linker monomer and residues thereof can be of varying lengths, such as from 1 to 25 atoms in length. For example, a linker monomer or residue can be from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 atoms in length, where any of the stated values can form an upper and/or lower end point. Further, the linker monomer or residue thereof can be substituted or unsubstituted. When substituted, the linker monomer or residue thereof can contain substituents attached to the backbone of the linker or substituents embedded in the backbone of the linker monomer or residue. For example, an amine substituted linker monomer or residue can contain an amine group attached to the backbone of the linker monomer or a nitrogen in the backbone of the linker monomer or residue. In some examples, the linker monomer and residues thereof can comprise a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol.

The linker monomer and residues thereof can, in further specific examples, comprise a substituted or unsubstituted Ci-C₆ branched or straight-chain alkyl, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, neo- pentyl, sec-pentyl, or hexyl. In a specific example, the linker monomer or residue thereof can comprise a polyalkylene (i.e., -(CH₂)_n-, wherein n is from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 1 to 5, from 1 to 3, or from 10 to 20). Still further, the linker monomer or residues thereof can comprise a cycloalkyl, such as cyclopentyl, cyclohexyl, or cyclopropyl. The linker monomer or residues thereof can also comprise a cycloheteroalkyl like piperazine, 2-methylpiperazine, l,3-di(piperidin-4-yl)propane, pyranyl, and the like. In another example, the linker monomer or residues thereof can comprise a Ci-C₆ branched or straight-chain alkoxy such as a methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso- butoxy, sec-butoxy, tert-butoxy, n-pentoxy, iso-pentoxy, neo-pentoxy, sec-pentoxy, or hexoxy. In still other examples, the linker monomer or residues thereof can comprise a C₂- C₆ branched or straight-chain alkyl, wherein one or more of the carbon atoms are substituted with oxygen (e.g., an ether), sulfur (e.g., a thioether), or nitrogen (e.g., an amino). For example, a suitable linker monomer or residue thereof can comprise a methoxymethyl, methoxyethyl, methoxypropyl, methoxybutyl, ethoxymethyl, ethoxyethyl, ethoxypropyl, propoxymethyl, propoxyethyl, methylaminomethyl, methylaminoethyl, methylaminopropyl, methylaminobutyl, ethylaminomethyl, ethylaminoethyl, ethylaminopropyl, propylaminomethyl, propylaminoethyl, methoxymethoxymethyl, ethoxymethoxymethyl, methoxyethoxymethyl, methoxymethoxyethyl, and the like, and derivatives thereof.

Y functional groups

The linker monomer can be any compound that can form a bond to two or more multi-sulfonamide monomers, linking them together. Thus a linker monomer typically contains at least two functional groups, e.g., one functional group that can be used to form a bond with one multi-sulfonamide monomer and another functional group that can be used to form a bond with another multi-sulfonamide monomer. These are shown as substituents Y¹ and Y² in Formula III. Alternatively, the linker monomer can have a functional group that is multivalent, i.e., a function group that can form more than one bond; that is, there can be additional Y substituents that are not shown in Formula III. Typically, though not necessarily, the functional group(s) on the linker monomer that is(are) used to form a bond with the multi-sulfonamide monomers are at opposite ends of the linker monomer. In some examples, the linker monomer can comprise nucleophilic functional groups (Y , Y², or both) that can react with electrophilic functional groups on the multi-sulfonamide monomer, forming a bond. Alternatively, the linker monomer can comprise electrophilic functional groups (Y¹, Y², or both) that can react with nucleophilic functional groups on the multi-sulfonamide monomer, forming a bond. Still further, the linker can comprise nucleophilic and electrophilic functional groups (Y , Y , or both) that can react with electrophilic and nucleophilic functional groups on the multi-sulfonamide monomer, forming a bond. The various arrangements are illustrated in the following table.

Table 1

Nucleophilic functional groups on the linker monomer

In certain examples, the linker monomer can comprise one or more nucleophilic functional groups that can react with one or more electrophilic functional groups on a multi- sulfonamide monomer to form a bond. Examples of nucleophilic functional groups that can be present on a linker monomer include, but are not limited to, an amine, amide, hydrazine, hydroxyl, or thiol group, as are described herein.

As noted, an example of a nucleophilic functional group that can be present on a linker monomer is an amine. In this instance, the amine acts as a nucleophilic functional group that can react with an electrophilic moiety on a multi-sulfonamide monomer {e.g., reacting with an alkenyl or alkynyl adjacent to an electron withdrawing group, halide, aldehyde, ketone, ester, acid anhydride, acid halide, or isocyanate to form an amine, imine, amide, or urea bond). For example, the linker monomer can comprise a primary amine {i.e., — NH₂), a secondary amine {i.e., — NHR²⁰), or a tertiary amine ( — NR²⁰ ₂), where each R²⁰ is, independent of the others, a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol. In some specific examples, the linker monomer can comprise a cyclic amine, such as aziridinyl, azetidinyl, pyrrolidinyl, pyrrolinyl, pyrazolidinyl, imidazolidinyl, pyrazolinyl, piperidinyl, piperazinyl, indolinyl, or morpholinyl group.

In one specific example, the linker monomer can comprise a polymeric amine, a polyamide, a polyether amine, a dendrimeric polyamine. Suitable examples of polymeric amines, polyamides, polyether amines, and dendrimeric polyamines are the same as those disclosed herein for the Z substituents.

In other similar examples, the linker monomer can comprise as a nucleophilic function group an amide {i.e., — NR²¹C(O)R²¹), where each R²¹ is, independent of the others, hydrogen, or is a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol.

Yet another suitable example of a nucleophilic group that can be present on a linker monomer is an alcohol. In this instance, the alcohol acts as a nucleophilic functional group that can react with an electrophilic moiety on a multi-sulfonamide monomer {e.g., reacting with an alkenyl or alkynyl adjacent to an electron withdrawing group, halide, aldehyde, ketone, ester, acid anhydride, acid halide, or isocyanate to form an ether, ester, acetal, carbonate, or urethane bond). For example, the linker monomer can be a primary alcohol, a secondary alcohol, or a tertiary alcohol, polymeric alcohol, or dendrimeric alcohol as disclosed above for the Z substituents.

Also, contemplated are linker monomers that comprise thiols, including primary, secondary, tertiary, dendrimeric, and polymeric thiols that correspond to the alcohols disclosed herein.

It is also contemplated that the linker monomer can contain more than one type of nucleophilic functional group. For example, the linker monomer can comprise an amine and an alcohol.

Electrophilic functional groups on the linker monomer

When the multi-sulfonamide monomer to be polymerized with the linker monomer comprises nucleophilic substituents, such as those listed above, the linker monomer can comprise an electrophilic or potentially electrophilic functional group. Examples of suitable electrophilic functional groups that can be used include, but are not limited to, an alkenyl or alkynyl moiety adjacent to an electron withdrawing group, halide, aldehyde, ketone, ester, acid anhydride, acid halide, or isocyanate groups. In one specific example, the suitable electrophilic functional group is an alkenyl or alkynyl moiety adjacent to a sulfonamide moiety.

In another examples, a suitable electrophilic functional group that can be present on a linker monomer includes, but is not limited to, aldehydes, acyl derivatives (e.g., acyl azides, acyl nitriles), esters and activated esters (e.g., succinimidyl esters, sulfosuccinimidyl esters), anhydrides and mixed anhydrides, derivatized carboxylic acids and carboxylates, imines, sulfonyl chlorides, organo-halides, and maleimides. In yet another example, a suitable electrophilic functional group that can be present on a linker monomer can be a polyester. Specific examples of polyesters are as disclosed above for the Z substituents. In still other examples, the linker monomer can comprise a halide, a ketone, an acid anhydride, or an acid halide moiety. These moieties are well known in the art of organic chemistry. Linker monomers containing succinimidyl ester moieties can also react with multi- sulfonamide monomers that contain amine, carboxylate, alcohol, or thiol functional groups. Succinimidyl ester containing linker monomers are particularly reactive towards amines, where the resulting amide bond that is formed is as stable as a peptide bond. However, some succinimidyl ester containing linker monomers may not be compatible with a specific application because they can be quite insoluble in aqueous solution. To overcome this limitation, sulfosuccinimidyl ester containing linker monomers, which typically have higher water solubility than succinimidyl ester containing linker monomers, can be used. Sulfosuccinimidyl ester containing linker monomers can generally be prepared in situ from simple carboxylic acid containing linker monomer by dissolving the linker monomer in an amine-free buffer that contains N-hydroxysulfosuccinimide and l-ethyl-3-(3- dimethylaminopropyl)carbodiimide. Also, 4-sulfo-2,3,5,6-tetrafluorophenol (STP) ester containing linker monomer can be prepared from 4-sulfo-2,3,5,6-tetrafluorophenol in the same way as sulfosuccinimidyl ester containing linker monomers.

In yet another example, the linker monomer can comprise an isocyanate moiety. Isocyanates are readily derivable from acyl azides, and they react with amine containing multi-sulfonamide monomers to form ureas, they react with alcohol containing multi- sulfonamide monomers to form urethanes, and they react with thiol containing multi- sulfonamide monomers to form thiourethanes.

Isothiocyanate moieties are an alternative to isocyanates and are moderately reactive but quite stable in water. Linker monomers containing isothiocyanates will react with an amine, alcohol, or thiol containing multi-sulfonamide monomer to form thioureas and thiourethanes.

Aldehyde containing linker monomers can react with nucleophilic substituents that contain amines to form Schiff bases. For example, a multi-sulfonamide monomer that comprises an amine can react with a dialdehyde containing linker like glutaraldehyde.

Organo-halide containing linker monomers contain a carbon atom bonded to a halide (e.g., fluorine, chlorine, bromine, or iodine). These moieties can react with multi- sulfonamide monomers that contain amine, carboxylate, alcohol, or thiol functional group to form, for example, amine, ester, ether, or sulfide bonds.

Also, when a linker monomer is not generally reactive it can be converted into a more reactive linker. For example, linker monomers that contain carboxylate or carboxylic acid groups can, depending on the conditions, be slow to react with a nucleophilic substituent on a multi-sulfonamide monomer. However, these linker monomers can be converted into more reactive, activated esters by a carbodiimide coupling with a suitable alcohol, e.g., 4-sulfo-2,3,5,6-tetrafluorophenol, N-hydroxysuccinimide or N- hydroxysulfosuccinimide. This results in a more reactive, water-soluble activated ester linking moiety. Various other activating reagents that can be used for the coupling reaction include, but are not limited to, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), N,N' -diisopropyl-carbodiimide (DIP), benzotriazol-1-yl- oxy-tris-(dimethylamino)phosphonium hexa-fluorophosphate (BOP), hydroxybenzotriazole (HOBt), and N-methylmorpholine (NMM), including mixtures thereof.

Specific Examples of linker monomers

Suitable linker monomers and residues thereof are readily commercially available and/or can be synthesized by those of ordinary skill in the art. And the particular linker monomer that can be used to prepare the disclosed sulfonamide-containing polymers can be chosen by one of ordinary skill in the art based on factors such as cost, convenience, availability, compatibility with various reaction conditions, the type of multi-sulfonamide monomer with which the linker monomer is to interact, and the like.

Some specific examples of suitable electrophilic linker monomers are dialdehydes and diesters, examples of which include, but are not limited to, gluteraldehyde, glyoxal, methylglyoxal, dimethyl-glyoxal, malonic dialdehyde, succinic dialdehyde, adipic dialdehyde, 2-hydroxyadipic dialdehyde, pimelic dialdehyde, suberic dialdehyde, azelaic dialdehyde, sebacic dialdehyde, maleic aldehyde, fumaric aldehyde, 1,3-benzenedialdehyde, phthalaldehyde, isophthalaldehyde, terephthalaldehyde, 1,4-diformylcyclohexane, and the like. Equivalents of dialdehydes that can be used instead of a dialdehyde include, 2,5- dialkoxytetrahydrofurans, 1,4-dialdehyde monoacetals, 1,4-dialdehyde diacetals. Examples of diesters include, but are not limited to, dialkyl oxylate, dialkyl fumarate, dialkyl malonate, dialkyl succinate, dialkyl adipate, dialkyl azelates, dialkyl suberate, dialkyl sebacate, dialkyl terephthalate, dialkylisophthalate, dialkylphthalate, and the like. Examples of diones include heptane-2,6-dione, hexane-2,5-dione, pentane-2,4-dione, and the like. Examples of diisocyanates include, but are not limited to, 1,3-phenyldiisocyanate, 1,4- phenyldiisocyanate, toluene diisocyanate, 1, 6-hexane-diisocyanate, and the like. Suitable linker monomers with nucleophilic functional groups capable of reacting directly or indirectly with an electrophilic Z substituent group on the multi-sulfonamide monomer include, but are not limited to, diamines, diols, dithiols, H₂N-(CH₂)_n-NH₂, (where n is some integer, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12), 2-pyridine disulfide, amino alcohols, amino thiols, compounds containing an alcohol and thiol, and the like.

Some specific examples of linker monomers that are suitable for use herein to prepare the disclosed sulfonamide-containing polymers are shown below in Formula IH-I through III-X.

where each R⁶ is, independent of the others, hydrogen, or is a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol; m is from 1 to 25; and Q is NR⁶, O, S, or CH₂; and each W is, independent of the others, N, CH or CR⁶.

Some still further specific examples of linker monomers that are suitable for use herein comprise an amine and/or alcohol functional groups are shown below as compounds 1-24.

7 8 9 10 11 12 13 I₅

Another suitable linker is compound 25:

.

It is also contemplated herein that the linker monomer may have two or more functional groups where one functional group reacts with and forms a bond to the multi- sulfonamide monomer and the other functional group(s) is available for further reaction with other compounds. For example, linker monomers 7-11, 15, and 23 have amine moieties that can react with the multi-sulfonamide monomer and hydroxyl groups that can be coupled to other molecules in order to alter the properties of the sulfonamide containing polymer. As a non-limiting example, the hydroxyl groups can be coupled to a saccharide (e.g., glucose, galactose, sucrose, fructose, cellulose), a protein (e.g., enzyme or antibody), or other biomolecule (e.g., folate, heparin, biotin, etc.). Such additional molecules can be coupled to the linker prior to the reaction with the multi-sulfonamide polymer or after polymerization of the multi-sulfonamide monomer and linker monomer to the sulfonamide- containing polymer

The polymerization reaction between the linker monomer and the multi-sulfonamide monomer results in a chemical bond that links the resulting linker residue to the multi- sulfonamide residue. As noted previously, such reactions can occur as a result of a direct nucleophilic or electrophilic interaction between the linker monomer and the multi- sulfonamide monomer. For example, a linker comprising a nucleophilic functional group can directly react with an electrophilic substituent on a multi-sulfonamide monomer and form a bond that links the resulting linker residue to the multi-sulfonamide residue. Alternatively, an electrophilic substituent on the linker monomer can directly react with a nucleophilic functional group on a multi-sulfonamide monomer and form a bond that links the resulting linker residue to the multi-sulfonamide residue. Also, the multi-sulfonamide monomer can be covalently attached to the linker monomer by an indirect interaction where a reagent initiates, mediates, or facilitates the reaction between the linker monomer and the multi-sulfonamide monomer. For example, the bond-forming reaction between the linker monomer and the multi-sulfonamide monomer can be facilitated by the use of a coupling reagent (e.g., carbodiimides, which are used in carbodiimide-mediated couplings) or enzymes (e.g., glutamine transferase).

When a linker monomer contains an amine functional group it can be particularly reactive toward multi-sulfonamide monomers with electrophilic functional groups. Such amine containing linker monomers can react with the multi-sulfonamide monomer and form, for example, depending on the functional groups of the multi-sulfonamide monomer, amine, amide, carboxamide, sulfonamide, urea, or thiourea bonds. When the linker monomer contains a carboxylate, they can react with electrophilic functional groups on the multi-sulfonamide monomer and form, for example, depending on the functional groups of the multi-sulfonamide monomer, esters, thioesters, carbonates, or mixed anhydrides. When the nucleophilic functional group on the linker monomer contains an alcohol or thiol, they can react with the functional group of the multi-sulfonamide monomer and form, for example, esters, thioesters, ethers, sulfides, disulfides, carbonates, or urethanes. In a specific example, a linker monomer comprising an amine functional group is reacted with a multi-sulfonamide monomer comprising as an electrophilic functional group an alkenyl or alkynyl adjacent to a carbonyl or sulfonamide (e.g., an α,/3-unsaturated carbonyl moiety or a vinyl sulfonamide moiety) in a Michael Addition reaction. Examples of such reactions are shown in Schemes 1-3.

Alternatively, when the multi-sulfonamide monomer contains an amine functional group, it can be particularly reactive toward a linker monomer comprising an electrophilic functional group. Such amine containing multi-sulfonamide monomers can react with the linker monomer and form, for example, depending on the electrophilic functional group on the linker monomer, amide, carboxamide, sulfonamide, urea, or thiourea bonds. When the nucleophilic multi-sulfonamide monomer contains a carboxylate, they can react with the linker monomer's electrophilic functional group and form, for example, esters, thioesters, carbonates, or mixed anhydrides. When the nucleophilic multi-sulfonamide monomer contains an alcohol or thiol, they can react with the linker monomer's electrophilic functional group and form, for example, depending on the substituent, esters, thioesters, ethers, sulfides, disulfides, carbonates, or urethanes.

The kinetics of such reactions depends on the reactivity and concentration of both the linker monomer and the multi-sulfonamide monomer. Also, factors affecting the reactivity of a linker monomer with an amine functional group are the amine 's class and basicity. For example, aliphatic amines are moderately basic and reactive with most electrophilic moieties. However, the concentration of the free base form of aliphatic amines below pH 8 is low; thus, the kinetics of a reaction between an aliphatic amine on an active substance and, for example, a vinyl sulfonamide moiety can be strongly pH dependent. While a pH of 8.5 to 9.5 is most efficient for attaching a multi-sulfonamide monomer with an electrophilic group to a linker monomer containing an amine, there will be some reactivity at pH 7 to pH 8.

Carbodiim ide-Mediated Coupling

In yet another example, a carbodiimide-mediated coupling can be used to form a bond between the linker monomer and the multi-sulfonamide monomer. For example, a linker monomer with an amine group can be coupled to an multi-sulfonamide monomer with a carboxylate or carboxylic acid functional groups using water-soluble carbodiimides such as l-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Suitable linker monomers capable of carbodiimide-mediate coupling to carboxylate or carboxylic acid containing multi- sulfonamide monomers are commercially available. Specific examples of carbodiimides include, but are not limited to, l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl, 1- cyclohexyl-3-(2-moφholinoethyl)-carbodiimide-metho-p-toluene sulfonate, and N,N'- dicyclohexylcarbodiimide.

In an alternative aspect involving a carbodiimide-mediated coupling, a linker monomer with a carboxylate or carboxylic acid group can be coupled to a multi- sulfonamide monomer with amine functional groups using water-soluble carbodiimides such as l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide.

Specific sulfonamide containing polymers

In certain examples, the disclosed sulfonamide-containing polymers can comprise residues of the multi-sulfonamide monomers with Formula A through G and residues of linker monomers with formula 1 through 24. Specific examples include sulfonamide- containing polymers wherein the polymer comprises repeating units of the following multi- sulfonamide residues and linker residues: Al, Bl, Cl, Dl, El, Fl, Gl, A2, B2, C2, D2, E2, F2, G2, A3, B3, C3, D3, E3, F3, G3, A4, B4, C4, D4, E4, F4, G4, A5, B5, C5, D5, E5, F5, G5, A6, B6, C6, D6, E6, F6, G6, A7, B7, C7, D7, E7, F7, G7, A8, B8, C8, D8, E8, F8, G8, A9, B9, C9, D9, E9, F9, G9, AlO, BlO, ClO, DlO, ElO, FlO, GlO, Al 1, Bl 1, Cl 1, Dl 1, El l, FI l, GI l, A12, B12, C12, D12, E12, F12, G12, A13, B13, C13, D13, E13, F13, G13, A14, B14, C14, D14, E14, F14, G14, A15, B15, C15, D15, E15, F15, G15, A16, B16, C16, D16, E16, F16, G16, A17, B17, C17, D17, E17, F17, G17, A18, B18, C18, D18, E18, F18, G18, A19, B19, C19, D19, E19, F19, G19, A20, B20, C20, D20, E20, F20, G20, A21, B21, C21, D21, E21, F21, G21, A22, B22, C22, D22, E22, F22, G22, A23, B23, C23, D23, E23, F23, G23, A24, B24, C24, D24, E24, F24, G24, A25, B25, C25, D25, E25, F25, and G25, including any mixtures thereof.

More specific examples include sulfonamide-containing polymers wherein the polymer comprises repeating units of the multi-sulfonamide residue T and linker residues: Tl, T2, T3, T4, T5, T6, T7, T8, T9, TlO, Tl 1, T12, T13, T14, T15, T16, T17, T18, T19, T20, T21, T22, T23, T24, and T25, including any mixtures thereof.

As noted elsewhere herein, the disclosed sulfonamide containing polymers can contain linker residues that can be further functionalized with other biomolecules and/or bioactive molecules. Such polymers are advantageous for combinatorial libraries geared towards screening a large number of compounds. In some examples, polymers containing hydroxyl groups can be reacted with saccharides (mono or poly) to form glycosides. Monosaccharides have been used in functionalizing polycationic polymers, such as polylysine, for improving gene delivery efficiency and targeting special cell types (Hashida et al., 2001 ; Hashida et al., 1998). The resulting polymers can also help to shield the positive charge on DNA nanoparticles, thereby hindering unwanted biological interactions and aggregation.

Folate-functionalized sulfonamide-containing polymers are also contemplated herein. Based on the studies by Hoekstra and co-workers, the route of uptake of plasmid DNA complexes strongly affects the efficiency of gene expression (Rejman et al., 2006). In particular, the internalization pathway of caveolae-mediated endocytosis appears to be particularly important for some polyplexes. As such, DNA particles with attached ligands that target caveolae (or lipid rafts) can show enhanced gene transfer efficiency. Anderson's group has shown that folate could be internalized efficiently via caveolae (Anderson, 1998; Anderson et al., 1992; Lee and Huang, 1996). Gene transfer vectors functionalized with folate, both lipidic and polymeric, have been studied for specifically targeting tumor cells (Lee and Huang, 1996; Ward et al, 2002). Thus, folate can be incorporated into the disclosed sulfonamide-containing polymers to target caveolae thus enhancing gene transfection efficiency. The synthesis of folate-functionalize polymers as disclosed herein can be executed as shown in the Scheme 2.

Targeting

In one aspect, the sulfonamide-containing polymers disclosed herein can also be coupled to a targeting moiety. For example, the multi-sulfonamide residue and/or linker residue can be attached to a targeting moiety. The targeting moiety can act to deliver or localize the sulfonamide-containing polymers to a particular area or particular cell type of a subject.

The sulfonamide-containing polymers disclosed herein may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al, Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al, Br. J. Cancer, 58:700-703, (1988); Senter, et al, Bioconjugate Chem., 4:3-9, (1993); Battelli, et al, Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al, Biochem. Pharmacol, 42:2062-2065, (1991)), all of which are herein incorporated by reference in their entirety for their teaching of the same. In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration.

Suitable targeting moieties can include an antibody or fragment thereof. The term "antibody" is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term "antibody" are antibody fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with a polypeptide of interest (e.g., diabodies, triabodies, and bi-specific and tri-specifϊc antibodies, as are known in the art; see, e.g. , Hudson and Kortt, J. Immunol. Methods 231:177-189, 1999), fusion proteins containing an antibody or antibody fragment, which are produced using standard molecular biology techniques, single chain antibodies, and human or humanized versions of immunoglobulin molecules or fragments thereof. Any antibody that specifically binds an antigen in a manner sufficient to target the sulfonamide containing polymers disclosed herein to the desired location can be used in the compositions and methods disclosed herein.

"Antibody fragments" are portions of a complete antibody. A complete antibody refers to an antibody having two complete light chains and two complete heavy chains. An antibody fragment lacks all or a portion of one or more of the chains. Examples of antibody fragments include, but are not limited to, half antibodies and fragments of half antibodies. A half antibody is composed of a single light chain and a single heavy chain. Half antibodies and half antibody fragments can be produced by reducing an antibody or antibody fragment having two light chains and two heavy chains. Such antibody fragments are referred to as reduced antibodies. Reduced antibodies have exposed and reactive sulfhydryl groups. These sulfhydryl groups can be used as reactive chemical groups or coupling of the sulfonamide containing polymers disclosed herein to the antibody fragment. A preferred half antibody fragment is a F(ab).

Antibody fragments for use in antibody conjugates can bind antigens. Preferably, the antibody fragment is specific for an antigen. An antibody or antibody fragment is specific for an antigen if it binds with significantly greater affinity to one epitope than to other epitopes. The antigen can be any molecule, compound, composition, or portion thereof to which an antibody fragment can bind.

Whenever possible, antibodies useful in the compositions and methods disclosed herein can be purchased from commercial sources, such as Chemicon International (Temecula, CA). The antibodies of the disclosed compositions and methods can also be generated using well-known methods. The skilled artisan will understand that either full- length antigens or fragments thereof can be used to generate the antibodies of the disclosed compositions and methods. A polypeptide to be used for generating an antibody of the disclosed compositions and methods can be partially or fully purified from a natural source, or can be produced using recombinant DNA techniques. For example, for antigens that are peptides or polypeptides, a cDNA encoding an antigen, or a fragment thereof, can be expressed in prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast, insect, or mammalian cells), after which the recombinant protein can be purified and used to generate a monoclonal or polyclonal antibody preparation that specifically binds the targeted antigen.

One of skill in the art will know how to choose an antigenic peptide for the generation of monoclonal or polyclonal antibodies that specifically bind the appropriate antigens. Antigenic peptides for use in generating the antibodies of the disclosed compositions and methods are chosen from non-helical regions of the protein that are hydrophilic. The PredictProtein Server or an analogous program can be used to select antigenic peptides to generate the antibodies of the disclosed compositions and methods. In one example, a peptide of about fifteen amino acids can be chosen and a peptide-antibody package can be obtained from a commercial source such as AnaSpec, Inc. (San Jose, CA). One of skill in the art will know that the generation of two or more different sets of monoclonal or polyclonal antibodies maximizes the likelihood of obtaining an antibody with the specificity and affinity required for its intended use. The antibodies can be tested for their desired activity by known methods (e.g., but not limited to, ELISA and/or immunocytochemistry). For additional guidance regarding the generation and testing of antibodies, see e.g., Harlow and Lane, Antibodies: A Labora tory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988, which is incorporated by reference herein at least for methods of making antibodies.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. Also disclosed are "chimeric" antibodies in which a portion of the heavy or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the ability to interact with a polypeptide of interest (See, U.S. Pat. No. 4,816,567 and Morrison et at, Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984) which are hereby incorporated by reference in their entirety for their taching of antibodies and uses thereof). Monoclonal antibodies useful in the compositions and methods disclosed herein can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature 1975, 256:495. In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro, e.g., using an adapter antigen or an immunogenic fragment thereof.

The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567 (Cabilly, et al, which is incorporated by reference herein at least for their methods of making antibodies). DNA encoding the monoclonal antibodies of the disclosed compositions can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 (Burton et al.) and U.S. Patent No. 6,096,441 (Barbas et al), which are incorporated by reference herein at least for their methods of making antibodies. Recombinant antibodies, antibody < fragments, and fusions and polymers thereof can be expressed in vitro or in prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast, insect, or mammalian cells) and further purified, as necessary, using well known methods (see e.g., Sambrook et al. Molecular Cloning: a Laboratory Manual, 3d Edition, Cold Spring Harbor Laboratory Press (2001); Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 2001, which are incorporated by reference herein at least for their methods of making antibodies).

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Patent No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen binding sites and is still capable of cross-linking antigen.

Any antibody or antibody fragment useful in the compositions and methods disclosed herein, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, e.g., to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment can be identified and/or improved by mutagenesis of a specific region of the nucleic acid encoding the antibody or antibody fragment, followed by expression and testing of the expressed polypeptide. For example, amino acid sequence variants of antibodies or antibody fragments can be generated and those that display equivalent or improved affinity for antigen can be identified using standard techniques and/or those described herein. Methods for generating amino acid sequence variants are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis or random mutagenesis (e.g., by PCR) of the nucleic acid encoding the antibody or antibody fragment (Zoller, Curr Opin Biotechnol 1992, 3:348-54). Both naturally occurring and non-naturally occurring amino acids (e.g., artificially- derivatized amino acids) can be used to generate amino acid sequence variants of the antibodies and antibody fragments used in the disclosed compositions.

As used herein, the term "antibody" or "antibodies" can also refer to a human antibody and/or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the compositions and methods disclosed herein serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

The human antibodies useful in the compositions and methods disclosed herein can be prepared using any technique. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an Fv, Fab, Fab', or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody. Further, examples of techniques for human monoclonal antibody production include those described by Cole, et al (Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boerner, et al (J Immunol 1991, 147(l):86-95). Human antibodies (and fragments thereof) useful in the compositions and methods disclosed herein can also be produced using phage display libraries (Hoogenboom et al, J MoI Biol 1991, 227:381; Marks et al, J MoI Biol 1991, 222:581; and Barbas, et al, Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). These references are incorporated by reference herein at least for their teaching of human antibody preparation.

The human antibodies useful in the compositions and methods disclosed herein can also be obtained from transgenic animals. For example, transgenic mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits, et al, Proc Natl Acad Sci USA 1993, 90:2551-5; Jakobovits et al, Nature 1993, 362:255-8; Bruggermann, et al, Year in Immunol 1993, 7:33, which are incorporated by reference herein at least for their teaching of human antibody preparation). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line transgenic mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge.

To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics {e.g., a certain level of specificity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies can also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones, et al, Nature 1986, 321 :522-5; Reichmann, et al, Nature 1988, 332:323-7; Presta, Curr Opin Struct Biol 1992, 2:593-6, which are incorporated by reference herein at least for their teachings of humanized antibodies).

Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated according to the methods of Winter and co-workers (Jones, et al, Nature 1986, 321:522-5; Riechmann, et al, Nature 1988, 332:323-7; Verhoeyen, et al, Science 1988, 239:1534-6), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567 (Cabilly et al), U.S. Patent No. 5,565,332 (Hoogenboom et al), U.S. Patent No. 5,721,367 (Kay et al), U.S. Patent No. 5,837,243 (Deo et al), U.S. Patent No. 5, 939,598 (Kucherlapati et al), U.S. Patent No. 6,130,364 (Jakobovits et al), and U.S. Patent No. 6,180,377 (Morgan et al). These references are incorporated by reference herein at least for their teachings of humanizing antibodies.

In addition to antibodies, other targeting moieties can be used to facilitate the targeting of the sulfonamide containing polymers disclosed herein to the appropriate cell type or area. Biomolecules

The disclosed sulfonamide-containing polymers also comprise a biomolecule. The biomolecule can be a polypeptide, peptidomimetic, nucleic acid, or a small molecule. For example, a biomolecule can be a protein, an oligosaccharide, a polysaccharide, DNA, RNA, siRNA, mRNA, microRNA, or a vitamin.

Nucleic Acids

As described above, the biomolecule of the disclosed sulfonamide-containing polymers can be nucleic acid based, including for example a nucleic acid that encodes one or more proteins, for example one or more therapeutic proteins, as well as various functional nucleic acids. The disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes.

A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The base moiety of a nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil- 1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. A non-limiting example of a nucleotide would be 3'- AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and TVU as well as different purine or pyrrolidine bases, such as uracil-5-yl (.psi.), hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. A modified base includes but is not limited to 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional base modifications can be found for example in U.S. Pat. No. 3,687,808, Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain nucleotide analogs, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine can increase the stability of duplex formation. Often times base modifications can be combined with, for example, a sugar modification, such as 2'-O- methoxyethyl, to achieve unique properties such as increased duplex stability. There are numerous United States patents such as 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, which detail and describe a range of base modifications. Each of these patents is herein incorporated by reference.

Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety would include natural modifications of the ribose and deoxyribose as well as synthetic modifications. Sugar modifications include but are not limited to the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or 0-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio, alkyl or C₂ to Cio alkenyl and alkynyl. T sugar modiifcations also include but are not limited to -O[(CH₂)_n O]_m CH₃, - O(CH₂)_n OCH₃, -O(CH₂)_n NH₂, -O(CH₂)_n CH₃, -O(CH₂)_n -ONH₂, and -O(CH₂)_nON[(CH₂)_n CH₃)]₂, where n and m are from 1 to about 10.

Other modifications at the T position include but are not limted to: Ci to Qo lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Modified sugars would also include those that contain modifications at the bridging ring oxygen, such as CH₂ and S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. There are numerous United States patents that teach the preparation of such modified sugar structures such as 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety. ^•■

Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. It is understood that this phosphate or modified phosphate linkage between two nucleotides can be through a 3'-5' linkage or a 2'-5' linkage, and the linkage can contain inverted polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Numerous United States patents teach how to make and use nucleotides containing modified phosphates and include but are not limited to, 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference.

It is understood that nucleotide analogs may not only contain a single modification, but may also contain multiple modifications within one of the moieties or between different moieties.

Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson- Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.

Nucleotide substitutes are nucleotides or nucleotide analogs that have had the phosphate moiety and/or sugar moieties replaced. Nucleotide substitutes do not contain a standard phosphorus atom. Substitutes for the phosphate can be for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones;formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Numerous United States patents disclose how to make and use these types of phosphate replacements and include but are not limited to 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.

It is also understood in a nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA). United States patents 5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is herein incorporated by reference. (See also Nielsen et al, Science, 1991, 254, 1497-1500). It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et ah, Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et ah, Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et ah, Ann. N.Y. Acad. Sci.,

1992, 660, 306-309; Manoharan et ah, Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et ah, Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et ah, EMBO J., 1991, 10, 1111-1118; Kabanov et ah, FEBS Lett., 1990, 259, 327-330; Svinarchuk et ah, Biochimie,

1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l^-di-O-hexadecyl-rac-glycero-S-H-phosphonate (Manoharan et ah, Tetrahedron Lett., 1995, 36, 3651-3654; Shea et ah, Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et ah, Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et ah, Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et ah, Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et ah, J. Pharmacol. Exp. Ther., 1996, 277, 923-937.

Numerous United States patents teach the preparation of such conjugates and include, but are not limited to U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.

A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute. A Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides.

Vectors

The biomolecule of the disclosed sulfonamide-containing polymers can be in a circularized or a linear form. For example, the biomolecules of the disclosed sulfonamide- containing polymers can be a vector or an expression vector comprising a polynucleotide of interest described elsewhere herein. For example, disclosed are sulfonamide-containing polymers and one or more of a biomolecule and a bioactive molecule, wherein the sulfonamide-containing polymer comprises Formula I:

— [— [multi-sulf onamidc] — [linker]—] — n Formula I wherein the multi-sulfonamide component is a residue of a multi-sulfonamide monomer that contains two or more sulfonamide moieties, and the linker component is a residue of a linker monomer and links one multi-sulfonamide residue to another, and n is from 2 to 10,000, wherein the biomolecule is an expression vector. Also disclosed herein are host cells transformed or transfected with an expression vector using the methods described elsewhere herein. Host cells can be eukayotic or prokaryotic cells.

Expression vectors can be any nucleotide construction used to express genes within cells (e.g., a plasmid). Expression vectors may also be used to express functional RNA molecules within cells, including but not limited to siRNAs, ribozymes, RNA aptamers and RNAi constructs. These expression vectors may be designed to integrate into specific integration sites found in cellular nucleic acid sequences, including but not limited to phage C31 integrase and attB sites, or may comprise sequences that mediate episomal replication of the expression vector, including but not limited to an S/MAR sequence.

The "control elements" present in an expression vector are those non-translated regions of the vector—enhancers, promoters, 5' and 3' untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the pBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or pSPORTl plasmid (Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.

Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters (e.g. beta actin promoter). The early and late promoters of the SV40 virus can be conveniently obtained as a restriction fragment, which also contains the SV40 viral origin of replication (Fiers et al, Nature, 273: 113 (1978)). The immediate early promoter of the human cytomegalovirus can be conveniently obtained as a HindIII E restriction fragment (Greenway, PJ. et al, Gene 18: 355-360 (1982)). Additionally, promoters from the host cell or related species can be used.

Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al, Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, MX., et al, MoI. Cell Bio. 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al, Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al, MoI. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many mammalian gene enhancer sequences are known(globin, elastase, albumin, α-fetoprotein and insulin), typically an enhancer from a eukaryotic cell virus will be used for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyomavirus enhancer on the late side of the replication origin, and adenovirus enhancers.

The promotor or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs. Optionally, the promoter or enhancer region can act as a constitutive promoter or enhancer to maximize expression of a polynucleotide of interest. In certain constructs the promoter or enhancer region can be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTR.

It is understood and herein contemplated that uncontrolled expression of a protein can have negative effects in a subject. As such, tissue specific promoters, cell type specific promoters, cell specific promoters, and/or a cell lineage specific promoters can also be used. For example, promoters from the host cell or related species are useful herein, and can be used for tissue specific gene expression or tissues specific regulated gene expression. "Tissue specific promoters" are promoters that are meant to only function in the environment created in a particular tissue. For example, tissue specific promoters can be used to target specific organs, tissue, grafts, or cells.

Examples of tissue specific promoters are known to those of skill in the art for most tissues (see e.g., WO 91/02805; EP 0,415,731; and WO 90/07936). Examples of suitable tissue specific promoters include, but are not limited to, neural specific enolase promoter, platelet derived growth factor beta promoter, human alpha 1-chimaerin promoter, synapsin I promoter, and synapsin II promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA. The 3¹ untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like endogenous mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases.

The expression vectors can include a nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Preferred marker genes are the E. coli lacZ gene, which encodes β-galactosidase, and the gene encoding green fluorescent protein. In some embodiments the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on the metabolism of a cell and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are CHO DHFR- cells and mouse LTK- cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which are not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al, MoI. Cell. Biol. 5: 410-413 (1985)). The three examples employ bacterial genes under the control of eukaryotic expression sequences to convey resistance to the appropriate drug neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Other selection drugs include the neomycin analog G418 and puramycin.

The biomolecules of the disclosed sulfonamide-containing polymers can also be expression vectors that are capable of integration into a mammalian chromosome without substantial toxicity.

The nucleic acids, such as, the polynucleotides described herein, can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 3rd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer. Synthetic methods useful for making oligonucleotides are also described by Ikuta et al, Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triester methods), and Narang et al, Methods Enzymol., 65:610-620 (1980), (phosphotriester method). Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et al, Bioconjug. Chem. 5:3-7 (1994).

Functional Nucleic Acids

As described above, the biomolecule of the disclosed sulfonamide-containing polymers can also be functional nucleic acids. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction. Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting. For example, functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, and external guide sequences. The functional nucleic acid molecules can act as affectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.

Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functional nucleic acids can interact with the mRNA of a molecule of interest or the genomic DNA of a molecule of interest or they can interact with a polypeptide of interest. Often functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.

Antisense molecules

The biomolecule of the disclosed sulfonamide-containing polymers can also be antisense molecules. Antisense molecules are designed to interact with a molecule of interest through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAse H mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC. It is preferred that antisense molecules bind the target molecule with a dissociation constant (k_<i) less than or equal to 10^~6, 10^"8, 10^"10, or 10^"12. A representative sample of methods and techniques which aid in the design and use of antisense molecules can be found in the following non-limiting list of United States patents: 5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437.

Aptamers

The biomolecule of the disclosed sulfonamide-containing polymers can also be aptamers. Aptamers are molecules that interact with a molecule of interest, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G- quartets. Aptamers can bind small molecules, such as ATP (United States patent 5,631,146) and theophiline (United States patent 5,580,737), as well as large molecules, such as reverse transcriptase (United States patent 5,786,462) and thrombin (United States patent 5,543,293). Aptamers can bind very tightly with k_<jS from the target molecule of less than 10^"12 M. It is preferred that the aptamers bind the target molecule with a ka less than 10^"6, 10^"8, 10^"10, or 10^"12. Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10,000 fold difference in binding affinities between the target molecule and other molecules that differ at only a single position on the molecule (United States patent 5,543,293). It is preferred that the aptamer have a ka with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the kj with a background binding molecule. It is preferred when doing the comparison for a polypeptide, for example, that the background molecule be a different polypeptide. Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,476,766, 5,503,978, 5,631,146, 5,731,424 , 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660 , 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698.

Ribozyntes

The biomolecule of the disclosed sulfonamide-containing polymers can also be a ribozyme. Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid molecules. It is preferred that the ribozymes catalyze intermolecular reactions. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase-type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes (for example, but not limited to the following United States patents: 5,334,711, 5,436,330, 5,616,466, 5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO 9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO 9718312 by Ludwig and Sproat), hairpin ribozymes (for example, but not limited to the following United States patents: 5,631,115, 5,646,031, 5,683,902, 5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), and tetrahymena ribozymes (for example, but not limited to the following United States patents: 5,595,873 and 5,652,107). There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (for example, but not limited to the following United States patents: 5,580,967, 5,688,670, 5,807,718, and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in the following non-limiting list of United States patents: 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855, 5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756.

Triplex forming functional nucleic acid molecules

The biomolecule of the disclosed sulfonamide-containing polymers can also be a triplex forming a functional nucleic acid molecule. Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid, for example a gene of interest. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a k_d less than 10^"6, 10^"8, 10^~10, or 10^"12. Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566, and 5,962,426.

External Guide Sequences

The biomolecule of the disclosed sulfonamide-containing polymers can also comprise external guide sequences. External guide sequences (EGSs) are molecules that bind a molecule of interest forming a complex, and this complex is recognized by RNAse P, which cleaves the molecule of interest. EGSs can be designed to specifically target a RNA molecule of interest. RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 by Yale, and Forster and Airman, Science 238:407-409 (1990)).

Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized as biomolecules to cleave desired molecules of interest within eukarotic cells. (Yuan et al, Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 by Yale; WO 95/24489 by Yale; Yuan and Airman, EMBO J 14:159-168 (1995), and Carrara et al, Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)). Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,168,053, 5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162.

PNAs

The biomolecule of the disclosed sulfonamide-containing polymers can also be peptide nucleic acids (PNAs). PNA is a DNA mimic in which the nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, Antisense Nucleic Acid Drug Dev. 1997; 7(4) 431-37). PNA can be utilized in a number of methods that traditionally have used RNA or DNA. Often PNA sequences perform better in techniques than the corresponding RNA or DNA sequences and have utilities that are not inherent to RNA or DNA. A review of PNA including methods of making, characteristics of, and methods of using, is provided by Corey (Trends Biotechnol 1997 June; 15(6):224-9). As such, in certain embodiments, one may prepare PNA sequences that are complementary to one or more portions of an mRNA sequence based on the molecule of interest, and such PNA compositions may be used to regulate, alter, decrease, or reduce the translation of the molecule of interest's transcribed mRNA, and thereby alter the level of the molecule of interest's activity in a host cell to which such PNA compositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester backbone of DNA (Nielsen et al, Science Dec. 6, 1991; 254(5037): 1497-500; Hanvey et al, Science. Nov. 27, 1992; 258(5087): 1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996 January; 4(l):5-23). This chemistry has three important consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAs are neutral molecules; secondly, PNAs are achirial, which avoids the need to develop a stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc or Fmoc protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used.

PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols (Norton et al, Bioorg Med Chem. 1995 April; 3(4):437-45). The manual protocol lends itself to the production of chemically modified PNAs or the simultaneous synthesis of families of closely related PNAs.

As with peptide synthesis, the success of a particular PNA synthesis will depend on the properties of the chosen sequence. For example, while in theory PNAs can incorporate any combination of nucleotide bases, the presence of adjacent purines can lead to deletions of one or more residues in the product. In expectation of this difficulty, it is suggested that, in producing PNAs with adjacent purines, one should repeat the coupling of residues likely to be added inefficiently. This should be followed by the purification of PNAs by reverse- phase high-pressure liquid chromatography, providing yields and purity of product similar to those observed during the synthesis of peptides.

Modifications of PNAs for a given application may be accomplished by coupling amino acids during solid-phase synthesis or by attaching compounds that contain a carboxylic acid group to the exposed N-terminal amine. Alternatively, PNAs can be modified after synthesis by coupling to an introduced lysine or cysteine. The ease with which PNAs can be modified facilitates optimization for better solubility or for specific functional requirements. Once synthesized, the identity of PNAs and their derivatives can be confirmed by mass spectrometry. Several studies have made and utilized modifications of PNAs (for example, Norton et al, Bioorg Med Chem. 1995 April; 3(4):437-45; Petersen et al, J Pept Sci. 1995 May- June; 1(3): 175-83; Orum et al, Biotechniques. 1995 September; 19(3):472-80; Footer et al, Biochemistry. Aug. 20, 1996; 35(33): 10673-9; Griffith et al, Nucleic Acids Res. Aug. 11, 1995; 23(15):3003-8; Pardridge et al, Proc Natl Acad Sci USA. Jun. 6, 1995; 92(12):5592-6; Boffa et al, Proc Natl Acad Sci USA. Mar. 14, 1995; 92(6):1901-5; Gambacorti-Passerini ef α/., Blood. Aug. 15, 1996; 88(4):1411-7; Armitage et al, Proc Natl Acad Sci USA. Nov. 11, 1997; 94(23): 12320-5; Seeger et al, Biotechniques. 1997 September; 23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA- DNA-PNA chimeric molecules and their uses in diagnostics, modulating protein in organisms, and treatment of conditions susceptible to therapeutics.

Methods of characterizing the antisense binding properties of PNAs are discussed in Rose (Anal Chem. Dec. 15, 1993; 65(24):3545-9) and Jensen et al (Biochemistry. Apr. 22, 1997; 36(16):5072-7). Rose uses capillary gel electrophoresis to determine binding of PNAs to their complementary oligonucleotide, measuring the relative binding kinetics and stoichiometry. Similar types of measurements were made by Jensen et al using BIAcore™ technology.

Other applications of PNAs that have been described and will be apparent to the skilled artisan include use in DNA strand invasion, antisense inhibition, mutational analysis, enhancers of transcription, nucleic acid purification, isolation of transcriptionally active genes, blocking of transcription factor binding, genome cleavage, biosensors, in situ hybridization, and the like.

Polypeptides

As described above, the biomolecules of the disclosed sulfonamide-containing polymers can be a polypeptide. As used herein, the term "polypeptide" is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post- expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. The polypeptide biomolecules can also contain insertions and/or deletions. Insertions include amino or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.

Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.

Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 2 and 3 and are referred to as conservative substitutions.

TABLE 2: Amino Acid Abbreviations

Amino Acid Abbreviations alanine Ala A arginine Arg R asparagine Asn N aspartic acid Asp D (aspartate) cysteine Cys C glutamic acid GIu E (glutamate) glutamine GIn Q glycine GIy G histidine His H isolelucine He I leucine Leu L lysine Lys K methionine Met M phenylalanine Phe F Amino Acid Abbreviations proline Pro P serine Ser S threonine Thr T tyrosine Tyr Y tryptophan Trp W valine VaI V

Glutamine or Gk Z Glutamic Acid

Any Amino Acid Xaa X

TABLE 3:Amino Acid Substitutions

Original Residue Exemplary Conservative Substitutions, others are known in the art. ala; ser arg; lys; gin asn; gin; his asp; glu cys; ser gin; asn; lys glu; asp gly; pro his; asn; gin ile; leu; val Leu; ile; val lys; arg; gin; Met; leu; ile phe; met; leu; tyr ser; thr thr; ser trp; tyr tyr; trp; phe val; ile; leu

Substantial changes in function are made by selecting substitutions that are less conservative than those in Tables 2 and 3, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, (c) the bulk of the side chain or (d) the integrity of binding pockets or active sites important for protein function. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl, (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation or glycosylation or (f) a residue required for protein function, e.g. an active site residue or a residue involved in binding an interacting partner, is substituted with any non-conservative residue.

For example, the replacement of one amino acid residue with another that is biologically and chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also may be desirable. Deletions or substitutions of potential proteolysis sites, e.g. Arg, are accomplished, for example, by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post- translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N- terminal amine and, in some instances, amidation of the C-terminal carboxyl.

Homology can be calculated using published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection. It is understood that the description of conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative mutations.

Biomolecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH₂NH-, -CH₂S-, -CH₂-CH₂ --, -CH=CH-- (cis and trans), -COCH₂ --, - CH(OH)CH₂-, and -CHH₂SO - (These and others can be found in Spatola, A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al, Int J Pept Prot Res 14:177-185 (1979) (-CH₂NH-, CH₂CH₂-); Spatola et al. Life Sci 38:1243-1249 (1986) (-CH H₂-S); Hann J. Chem. Soc Perkin Trans. I 307-314 (1982) (-CH-CH-, cis and trans); Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (-COCH₂-); Jennings-White et al. Tetrahedron Lett 23:2533 (1982) (-COCH₂-); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982) (-- CH(OH)CH₂-); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (--C(OH)CH₂-); and Hruby Life Sci 31 : 189- 199 ( 1982) (-CH₂-S-); each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is -CH₂NH-. It is understood that peptide analogs can have more than one atom between the bond atoms, such as β-alanine, γ- aminobutyric acid, and the like.

Amino acid analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.

D-amino acids can be used to generate more stable peptides, because D-amino acids are not recognized by peptidases and such. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations. (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference).

Antibodies

As described above, the biomolecule of the disclosed sulfonamide-containing polymers can be an isolated antibody, antibody fragment and/or antigen-binding fragments thereof. Optionally, the isolated antibodies, antibody fragments, or antigen-binding fragment thereof can be neutralizing antibodies. Antibodies and there general structure and function are described above. Additionally, the antibodies, antibody fragments and antigen- binding fragments thereof disclosed herein can be identified using the methods disclosed herein. Administration of Compounds

The disclosed sulfonamide-containing polymers can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, by intravitreal delivery, by intraotic delivery, by injection directly to the desired area of action, transdermally, extracorporeally, by delivery directly to the cerebral spinal fluid (CSF), including intrathecal injection, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular sulfonamide-containing polymer used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated herein by reference in its entirety for its teaching of an approach for parenteral administration.

The sulfonamide-containing polymers may be in solution or microparticle suspension.

It will be understood that, if desired, a sulfonamide-containing polymer as disclosed herein may be administered in combination with other agents as well, such as, e.g., proteins, polypeptides or various pharmaceutically-active agents. In fact, there is virtually no limit to other components that may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The compositions may thus be delivered along with various other agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein. Likewise, such compositions may further comprise substituted or derivatized RNA or DNA compositions.

Also disclosed herein are pharmaceutical compositions comprising any of the compositions disclosed herein and a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, Ringer's solution, dextrose solution, and buffered solutions at physiological pH. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the sulfonamide-containing polymers, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Other compounds will be administered according to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the compositions disclosed herein. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed sulfonamide-containing polymers can be administered intravenously, intravitreally, intraotically, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally or injected directly in the desired site of transfection.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are DMSO, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.

Some of the sulfonamide-containing polymers may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. Immunogenic Compositions

Also disclosed are illustrative immunogenic compositions, e.g., vaccine compositions, that comprise DNA encoding a polypeptides as described above, such that the polypeptide is generated in situ. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143- 198, 1998, and references cited therein, all of which are herein incorporated by reference in their entirety for their teaching of gene delivery techniques. Appropriate polynucleotide expression systems contain the necessary regulatory DNA regulatory sequences for expression in a subject (such as a suitable promoter and terminating signal), as described above.

Additionally, the pharmaceutical compositions described herein can comprise one or more immunostimulants in addition to the compositions of this invention. An immunostimulant refers to essentially any substance that enhances or potentiates an immune response (antibody or cell-mediated) to an exogenous antigen. One preferred type of immunostimulant comprises an adjuvant. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Certain adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Rahway, NJ.); AS-2 (GlaxoSmithKline, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and.quil A. Cytokines, such as GM- CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.

The adjuvant composition can be a composition that induces an anti-inflammatory immune response (antibody or cell-mediated). Accordingly, the adjuvant composition can comprise high levels of anti-inflammatory cytokines (anti-inflammatory cytokines may include, but are not limited to, interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 10 (IL- 10), and transforming growth factor beta (TGFjS)). Optionally, an anti-inflammatory response would be mediated by CD4+ T helper cells. Bacterial flagellin has been shown to have adjuvant activity (McSorley et ah, J. Immunol. 169:3914-19, 2002). Also disclosed are polypeptide sequences that encode flagellin proteins that can be used in adjuvant compositions.

Optionally, the adjuvants used in conjunction with the compositions of the present invention increase lipopolysaccharide (LPS) responsiveness. Illustrative adjuvants include but are not limited to, monophosphoryl lipid A (MPL), aminoalkyl glucosaminide 4- phosphates (AGPs), including, but not limited to RC-512, RC-522, RC-527, RC-529, RC- 544, and RC-560 (Corixa, Hamilton, Mont.) and other AGPs such as those described in pending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720, the disclosures of which are incorporated herein by reference in their entireties. In addition, the adjuvant composition can be one that induces an immune response predominantly of the ThI type. High levels of Thl-type cytokines (e.g., IFN-γ, TNFα, IL-2 and IL- 12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-IO) tend to favor the induction of humoral immune responses. Following application of a immunogenic composition as provided herein, a subject will support an immune response that includes ThI- and Th2-type responses. Optionally, the level of Thl- type cytokines can be increased to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989, which is hereby incorporated by reference for its teaching of families of cytokines. The level of Th2-type cytokines can be increased to a greater extent than the level of Thl- type cytokines.

Certain adjuvants for eliciting a predominantly Thl-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with aluminum salt adjuvants are available from Corixa Corporation (Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094, which are hereby incorporated by reference for their teaching of the same). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly ThI response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al, Science 273:352, 1996. Another adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins. Other formulations can include more than one saponin in the adjuvant combinations of the present invention, for example combinations of at least two of the following group comprising QS21, QS7, Quil A, /3-escin, or digitonin.

Saponin formulations can also be combined with vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc. The saponins can also be formulated in the presence of cholesterol to form particulate structures such as liposomes or immune-stimulating complexes (ISCOMs). Furthermore, the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM. The saponins can also be formulated with excipients such as CARBOPOL ^M (Noveon, Cleveland, Ohio) to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose.

Optionally, the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and 3D-MPL adjuvant, as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other formulations comprise an oil-in-water emulsion and tocopherol. Another adjuvant formulation employing QS21, 3D-MPL.RTM. adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

Another enhanced adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 is disclosed in WO 00/09159. Optionally the formulation additionally comprises an oil in water emulsion and tocopherol.

Additional illustrative adjuvants for use in the immunogenic compositions of the invention include Montamide ISA 720 (Seppic, France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants {e.g., SBAS-2 or SBAS-4, available from GlaxoSmithKline, Philadelphia, Pa.), Detox (Enhanzyn™) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720, the disclosures of which are incorporated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549A1.

Effective Dosage

Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms and/or the disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the subject, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

Following administration of a disclosed composition for treating, inhibiting, or preventing a disease, the efficacy of the therapeutic composition can be assessed in various ways well known to the skilled practitioner.

The disclosed compositions and methods can also be used for example as tools to isolate and test new drug candidates for a variety of diseases. Methods

The disclosed compositions can be used in a variety of different methods, for example in prognostic, predictive, diagnostic, and therapeutic methods and as a variety of different compositions. The disclosed compositions can also be used as research reagents. For example, disclosed herein are methods that utilize sulfonamide-containing polymers for gene transfection and delivery of other biomolecules. Illustrating this ability, disclosed herein are methods of introducing a biomolecule to a subject comprising administering one or more of the disclosed compositions to the subject. Subjects that can be used in this and the other methods described below are described above. In addition, biomolecules are defined and described above as well. The methods below can be used for gene therapy or> other forms of genetic manipulation of a subject of cell.

Also disclosed herein are methods of bringing into contact a biomolecule and a cell comprising contacting the cell with one or more of the disclosed compositions, thereby bringing into contact the biomolecule and the cell.

Also disclosed herein are methods of modulating a molecule of interest in a cell comprising contacting a cell with one or more of the disclosed compositions under conditions that allow the biomolecule of the composition to modulate the molecule of interest, wherein the molecule of interest is modulated. The cell can be in a subject or in culture. As such, the method can be performed in vivo, in vitro, or ex vivo. Molecules of interest are defined and described above. For example a molecule of interest can be a nucleic acid or protein. As such, the described methods can be used to reduce, increase, enhance, or restore the expression or activity of a molecule of interest.

Also described herein are methods of modulating the expression or activity of a biomolecule of interest in a subject comprising administering one or more of the disclosed compositions to the subject under conditions that allow the biomolecule of the composition to modulate the expression or activity of the biomolecule of interest, wherein the expression or activity of a biomolecule of interest is modulated.

The methods and compositions disclosed herein can also be used in the prevention, amelioration, or treatment of medical disorders, such as those known to those of skill in the art. For example, the disclosed compositions can be used for gene therapy, where the disclosed compostions comprise a nucleic acid as the biomolecule. Additionally, described herein are methods of treating a subject comprising administering a therapeutically effective amount of one or more of the disclosed compositions to the subject in need thereof.

The disclosed compositions can also be used for screening purposes. For example, described herein are methods of screening for a biomolecule that modulates a molecule of interest in a cell, comprising introducing to a cell comprising a molecule of interest one or more of the disclosed compositions, wherein the composition comprises the biomolecule, determining the expression or activity of the molecule of interest compared to a control, wherein a change in activity of the molecule of interest indicates a biomolecule that modulates the molecule of interest.

Furthermore, described herein are methods of screening for the effect of a biomolecule in a cell comprising introducing a biomolecule to a cell comprising administering one or more of the disclosed compositions, determining the effect of the biomolecule compared to a control, wherein a change from the control indicates the effect of the biomolecule.

Also described herein are methods of screening for additional sulfonamide- containing polymers to be used in the disclosed methods. For example, disclosed herein are methods of screening for sulfonamide-containing polymers capable of delivering a biomolecule to a cell comprising administering a sulfonamide-containing polymer comprising a biomolecule, determining whether the biomolecule was delivered to the cell compared to a control, wherein a change from the control indicates the ability of the sulfonamide-containing polymer to deliver the biomolecule to a cell.

In the screening methods disclosed above, the screening can be high throughput. In addition, the screening methods disclosed herein can be performed in vitro, in vivo, or ex vivo. For example, the screeing methods can be carried out as described in the Examples below.

The compositions disclosed herein can also be used to transform plants. The present invention thus provides methods for genetically engineering plants to provide inventive transformed plants which may be readily delignified. The invention can feature the disclosed compositions further comprising a tissue-specific plant promoter sequence, as well as DNA constructs comprising nucleotide sequences having substantial identity thereto and having similar levels of functionality. The disclosed compostions can be used to produce a recombinant DNA expression system, which is also an aspect of the invention. Kits

Disclosed herein are kits that can be used in practicing the methods disclosed herein. The kits can include any reagent or combination of reagents discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods. For example, the kits could include one or more of the disclosed compositions. The disclosed kits could optionally comprise a pharmaceutical carrier. The disclosed kits could further optionally comprise an expression vector.

The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples. Rather, in view of the present disclosure that describes the current best mode for practicing the invention, many modifications and variations would present themselves to those of skill in the art without departing from the scope and spirit of this invention. All changes, modifications, and variations coming within the meaning and range of equivalency of the claims are to be considered within their scope.

EXAMPLES

The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, pH, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ⁰C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of conditions, e.g., component concentrations, temperatures, pressures, and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions. Example 1 Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compositions are either available from commercial suppliers such as Ocean Nutrition Canada, Ltd. (Dartmouth, NS, Canada), Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplements (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

Key disadvantages of nonviral vectors are cytotoxicity and low efficiency. As such, the first synthesis scheme focused on devising polymer systems with predicted biodegradability and low toxicity. In humans, sulfonamide moieties have been widely used in pharmaceuticals (antimicrobials, diuretics, hypoglycemic agents) and are likely to be well tolerated in animals (Rang et ah, 1995). In addition, a variety of poly(ester-amines) and poly(amido-amines) have been used in biomedical applications including gene delivery (Anderson et al, 2003; Ferruti et ah, 1994). The first scheme draws on both groups of compounds to create a new class of gene transfer polymers based on poly(beta- aminosulfonamides), abbreviated PBASs, that are predicted to be biodegradable with reduced cytotoxicity. (See Figures 1 and 2). Synthesis

Polymerization of divinylsulfonamides with bis(secondary amines), bis(primary amines) and mono(primary amine) utilizing the Michael addition reaction afforded a series of poly(/3-aminosulfonamides) (Figures 1 and 2). Divinyl sulfonamide monomers A-G and amine monomers 1-24 were chosen for the synthesis of a screening library for transfection studies. All chemicals were purchased from commercial suppliers and used without further purification.

Monomer Synthesis

A general procedure for the synthesis of divinyl sulfonamide monomers having Formulae A-G is provided using monomer A as an example. To a solution of /J- chloroethanesulfonylchloride (42 mmol) in 50 mL of dry dichloromethane, a solution of 2- methylpiperazine (20 mmol) and triethylamine (85 mmol) in 50 mL of dry dichloromethane was added dropwise at 0 °C under nitrogen flow. After addition, the reaction mixture was stirred magnetically for 2 hours at 0 ⁰C and overnight at room temperature. The solid formed was filtered and the filtrate was washed with water, brine and then water. The organic layer was dried over dry sodium sulfate and concentrated. Pure product was obtained after column chromatography (a mixture of dichloromethane and ethyl acetate as eluent) as a pale yellow solid or liquid. Solid monomers were further purified by recrystallization from a mixture of diethyl ether and dichloromethane (Yield: 30 - 60 %). ¹H NMR spectra were recorded at 400 or 500 MHz (¹³C spectra at 100 or 125 MHz) on Bruker DRX-400 or DRX-500 spectrometers, respectively. Spectral characterization for monomers A-G is as follows.

¹H NMR (500 MHz, CDCl₃): δ 6.38 - 6.44 (two dd, overlapping, 2H), 6.27 (d, J= 16.5 Hz, IH), 6.24 (d, J= 16.5 Hz, 1H),6.O7 (d, J= 10.0 Hz, IH), 6.00 (d, J= 10.0 Hz, IH), 4.18 (m, IH), 3.68 (d, J = 11.7 Hz, IH), 3.59 (d, J = 11.7 Hz, IH), 3.50 (d, J = 11.7 Hz, IH), 3.25 (m, IH), 2.89 (m, IH), 2.71 (m, IH), 1.35 (d, J=6.8 Hz, 3H). ¹³C NMR (100 or 125 MHz, CDCl₃): δ 135.6, 132.2, 129.5, 127.4, 122.3, 50.89, 50.86, 48.55, 48.50, 45.7, 44.7, 39.5, 14.9. MS m/z 281.0.

¹H NMR (400 MHz, ₃): δ 6.42 (dd, 2H), 6.20 (d, J= 1 Hcz, 2H), .6.02 (d, J= 9.9 Hz, 2H), 3.70 (d, J= 12.0 Hz, 4H), 2.55 (t, J = 11.5 Hz, 4H), 1.75 (d, J=9.8 Hz, 4H), 1.21-1.27 (m, 12H). ¹³C NMR (100 or 125 MHz, CDCl₃): δ 132.8, 128.2, 46.1, 36.3, 35.4, 31.8, 23.7. MS m/z 391.1.

¹H NMR (400 MHz, CDCl₃): δ 6.42 (dd, 2H), 6.15 (d, J= 16.5 Hz, 2H), 5.95 (d, J= 9.8 Hz, 2H), 322 (s, 4H), 2.77 (s, 6H). ¹³C NMR (100 or 125 MHz, CDCl₃): δ 132.9, 127.9, 48.1, 34.9. MS m/z 269.0.

¹H NMR (500 MHz, CDCl₃): δ 6.46 - 6.55 (two dd, overlapping, 2H), 6.26 (d, J= 16.4 Hz, IH), 6.25 (d, J= 16.4 Hz, 1H),5.98 (d, J= 10.1 Hz, IH), 5.92 (d, J= 10.1 Hz, IH), 4.99 (m, IH), 4.30 (m, IH), 4.21 (q, J = 7.4 Hz, 2H), 3.88 (m, IH), 3.00 (q, J = 6.6 Hz, 2H),1.85 (m, IH)₅1.43-1.71 (m, 5H), 1.29 (t, J=7.4 Hz, 3H). MS m/z 355.0.

¹H NMR (400 MHz, CDCl₃): δ 6.42 (dd, 2H), 6.22 (d, J= 16.7 Hz, 2H), 5.98 (d, J= 9.9 Hz, 2H), 3.07 (t, J=3.5 Hz, 4H), 2.77 (s, 6H), 1.58 (m, 6H), 1.38 (m, 4H). ¹³C NMR (100 or 125 MHz, CDCl₃): δ 132.8, 128.2, 46.1, 36.3, 35.4, 31.8, 23.7. MS m/z 325.0.

¹H NMR (400 MHz, CDCl₃): δ 6.58 (dd, 2H), 6.23 (d, J= 16.7 Hz, 2H), 5.96 (d, J= 9.9 Hz, 2H), 5.30 (s, 2H), 3.63 (m, 8H, overlapping), 3.19 (t, J= 6 Hz, 4H). MS m/z 663.4.

¹H NMR (400 MHz, CDCl₃): δ 6.43 (dd, 2H), 6.26 (d, J= 16.7 Hz, 2H), 6.11 (d, J= 9.9 Hz, 2H), 3.28 (s, 8H). ¹³C NMR (100 or 125 MHz, CDCl₃): δ 132.1, 129.7, 45.4, 45.3. MS m/z 267.0. Polymer Synthesis

Several model reactions were carried out to optimize the reaction conditions. Steric hindrance of both amine monomers and divinylsulfonamides plays a role in the Michael reaction. Chloroform was chosen as the solvent because of its good solubility for both monomers and resulting polymers. Isopropanol can accelerate the reaction by helping with proton transfer. No adduct product of isopropanol with divinylsulfonamides could be detected. For reactions that were sluggish, different Lewis acid catalysts were tested and LiClO₄ was found to accelerate the reaction drastically. LiClO₄ also has good solubility in the polymerization solvent system. Based on the model reactions, the typical polymerization condition was set as follows: Equal molar amounts (2.0 mmol) of amine monomers and divinylsulfonamides were weighed into vials equipped with Teflon coated stir bars, followed by 4.0 mmol OfLiClO₄, then 1 mL anhydrous chloroform and 1 mL IPA were added. After capping with Teflon lined screw caps, the vials were kept at 75 ⁰C in the dark for one week. Both bis(secondary amines) and mono(primary amine) monomers adopted the typical condition. For bis(primary amines), gelation was observed due to crosslinking, affording insoluble solids at the above condition. Thus, the reaction was carried out at room temperature in chloroform for 3 days in the dark. No gelation was observed for polymers prepared this way even after storage for two months. When polymerization was complete, the polymer was precipitated in diethyl ether. The precipitate was dissolved in DMSO, DMF, or chloroform followed by precipitation in diethyl ether or THF repeatedly. One of the advantages of using LiClO₄ as catalyst was that it has good solubility in diethyl ether (114 g/ 100 mL ether), which facilitates the purification process. The polymers were then dried under vacuum at 60 ⁰C for 2 days.

¹H NMR spectra were recorded at 400 or 500 MHz (¹³C spectra at 100 or 125 MHz) on Bruker DRX-400 or DRX-500 spectrometers, respectively. All the peaks in the ¹H NMR and ¹³C NMR spectra could be assigned. The signal of the vinyl group was weak, but still observed, indicating the degree of polymerization was not high.

MALDI-TOF MS was used to determine the molecular weights and polydispersity of the resulting polymers (Hanton, Chem. Rev. 101 :527-569, 2001). Shown in Figure 3 is a typical MALDI spectrum obtained. The molecular weights of these polymers ranged from 1000 to 8000 Da. High molecular weights are not necessary. From the expansion of the MALDI mass spectrum, the end groups could be determined, in this case, vinyl-vinyl, vinyl- amine or amine-amine end groups could be detected. Mass spectral data were collected in a linear mode. The ions are generated using the 337 nm laser beam from a nitrogen laser, having a pulse width of 3 ns. No correction of l/(dm/dt) (Pack et al, Nature Rev. Drug Discovery 4:581-593, 2005) was applied to the mass spectra during the conversion of the time domain to the mass domain. Average molecular weights (M_n, M_w) and PDI were determined directly from the time domain according to the following equations

M_n = ∑ (NMi) /∑ Ni M^ = 2 (M,Λφ/ ∑ N₁M_f

PDI = M_w / M_n where Mj is the mass of an observed ion and N; is the number of ions observed. The average molecular weights are calculated from Na⁺-cationized oligomers. The ion masses were corrected for the mass of the cation, substracting 23 u in this case. For compound E9, the characterization data is as follows.

¹H NMR (400 MHz, DMSO): δ [6.7 (dd, trace), 6.1 (d, trace), 6.0 (d, trace), 3.4 (2H), 3.1 (4H), 3.0 (4H), 2.8 (4H), 2.7 (6H), 2.4 (2H), 1.5 (4H), 1.4 (4H), 1.3 (4H)] ¹³C NMR (100 or 125 MHz, DMSO): δ 133.3 (trace), 127.8 (trace), 60.6, 52.3, 49.3, 46.6, 45.5, 34.2, 30.2, 27.3, 25.6, 23.3. MALDI MS M_w = 4600].

For compound E3, the characterization data is as follows.

MALDI MS M_w = 4500.

For compound E6, the characterization data is as follows:

MALDI MS M_w = 2200.

For compound E8, the characterization data is as follows:

MALDI MS M_w = 2400.

For compound E24, the characterization data is as follows:

MALDI MS Mw = 1600.

For compound G8, the characterization data is as follows:

MALDI MS M_w = 2300.

For compound C6, the characterization data is as follows:

MALDI MS M_w = 1600.

For compound C9, the characterization data is as follows:

MALDI MS M_w = 4000.

For compound ClO, the characterization data is as follows:

MALDI MS M_w = 3400.

For compound BI l, the characterization data is as follows:

MALDI MS Mw = 4100.

For compound A6, the characterization data is as follows:

MALDI MS M_w = 3900.

For compound Bl 1 -glucose, the characterization data is as follows:

¹H NMR (400 MHz, DMSO): δ 6.7 (dd, trace), 6.2 (0.06H), 6.1 (d, trace), 6.0 (d, trace), 4.9 (0.04H), 4.8 (0.05H), 4.6 (0.04H), 4.5 (0.05H), 4.3 (2H), 3.5 (6H), 3.4 (2H), 3.0-3.1 (6H), 2.7-2.9 (6H), 1.7 (4H), 1.0-1.4 (18H). MALDI MS M_w = 4200.

For compound E9-glucose, the characterization data is as follows:

¹H NMR (400 MHz, DMSO): δ 6.7 (dd, trace), 6.2 (0.36H), 6.1 (d, trace), 6.0 (d, trace), 4.9 (0.40H), 4.8 (0.38H), 4.6 (0.35H), 4.5 (0.50H), 4.3-4.4 (1.6H), 3.8 (0.36H), 3.5-3.6 (1.2H), 3.1 (4H), 3.0 (6H), 2.8 (4H), 2.7 (6H), 2.4 (2H), 1.5 (4H), 1.4 (4H), 1.2 (4H). MALDI MS M_w = 9900.

For compound T+l (nomenclature according to Figure 13), the characterization data is as follows: N

¹H NMR (400 MHz, DMSO): δ [7.8 (2H), 7.5 (IH), 7.3 (2H), 7.1 (IH), 6.9 (IH), 4.0 (4H), 3.9 (2H), 2.7 (6H), 2.4 (3H), 2.3 (6H), 2.2 (2H), 2.1 (4H), 1.8 (2H). MALDI MS M_w = 2900. For compound C 18, the characterization data is as follows:

MALDI MS M_w = 5200.

For compound E21, the characterization data is as follows:

MALDI MS M_w = 1700.

For compound G 13, the characterization data is as follows

MALDI MS MW = 1000

For compound C22, the characterization data is as follows

MALDI MS MW = 3000

For compound A7, the characterization data is as follows

MALDI MS MW = 3100

For compound C 15, the characterization data is as follows

MALDI MS MW = 1200 Acid-Base Titration Profile

Tertiary amines in the polymer back bones of poly(/3-aminosulfonamides) (PBAS) can impart pH-sensitive characteristics to the polymers. It can result in a proton buffering effect within the endosomal/lysosomal compartments of the cell. Therefore, acid-base titration of the polymers were performed (representative data shown in Figure 4). Both polymers and NaCl were dissolved in water and adjusted to pH 2 with 0.1 N HCl solution followed by titration with 0.1 N NaOH until pH 12. pH buffering region of E3 is 5.1-5.4, while that of E9 is 5.2-7.2.

Table 4: Molecular weights and pH buffer range of sulfonamide-containing polymers disclosed herein

Gel retardation Assay of Polyplexes

The polymers soluble in aqueous solution were used to form complexes with plasmid DNA (pDNA). Figure 5 shows the electrophoretic shifts for pDNA in the presence of different amounts of polymers. The results demonstrate that most PBAS polymers could bind with pDNA to form stable complexes at ratios larger than 1 :2. AFM studies

Atomic Force Microscopy (AFM) studies were performed to observe the morphology of the formed polyplexes. Figure 6 shows the AFM image of the pDNA only and polyplexes formed by mixing pDNA with polymer E3 at molar ratio of 1 :2. Spherical complexes were observed, and the diameters varied from 60 nm to 100 nm. After mixing the pDNA with polymer, no free pDNA could be detected by AFM, which is in agreement with the gel retardation assay.

Example 2

Direct electroporation has now emerged as the single best system for spatially and temporally restricted genetic manipulation in embryonic brains (Washbourne and McAllister, 2002). Like all techniques, however, in vivo electroporation has limitations: it is now clear that, for embryonic electroporation to be done well, with reproducible results and little or no tissue damage, months of practice and experience in the embryology of electrode placement are required for each experimenter.

Chemical transgenesis has not been much explored in whole animal studies even though it is the standard method of cell culture transfection and has attracted great interest from gene therapists. Commercial transfection reagents for chick neural tube transgenesis, which use lipids or cationic polymer carriers, were developed specifically for use in tissue culture and not for in vivo work. Indeed, every lipid and liposomal method tested (e.g., Lipofectamine, Life Technologies; GeneFECTOR, Venn Nova; DOTAP, Roche) yielded little or no transgenesis in chick embryos. Other groups have had similar experience (Muramatsu et al, 1997). Cationic polymers, including polyethylenimine (PEI) were also tested. Some of these reagents showed more promise as in ovo transfectants and led to the focus of the work described below on cationic polymer vectors rather than lipid-based compounds. In vitro Transfection Studies

All the polymers were tested in COS-7 cell luciferase transfection assays. The results indicated that the best in vitro transfectants are C6, C 18, E21 and E24 (Figure 7; Figure 15). They are almost 10 fold more efficient than JetPEI. The pH buffer range did not correlate well with the transfection efficiency in vitro as E24 and Gl 3 have a narrow pH range. The results may imply that the gene delivery pathway for these polymers may be different from those that involve so called "proton sponge" in endosome/lysosome. The detailed biological studies on the delivery pathway are important to elucidate the mechanism and are in the progress. It is also equally important to point out that efficient transfectants in vitro do not guarantee their success for in vivo delivery. These in vitro gene delivery results showed that several of the disclosed sulfonamide-containing polymers are very active transfectants and showed higher efficiencies than those from well known commercial sources simultaneously tested (Figure 7; Figure 15). In vivo Transfection Studies

Of the 57 compounds synthesized to date, 49 were soluble in water, pH 5 buffer or DMSO. These polymers were directly tested as in vivo transfectants in chick embryo midbrain injections using marker gene plasmids as reporters (Figure 8). Figure 8 shows representative data for the polyplex transfection efficacies studied in vivo. Chick embryo midbrain screens were carried out with the commercially available dendrimer SuperFect (A), the efficient PBAS polymers E9 (B) and E3/3900 Da (C), and the teratogen B17 (D). Embryonic day 5 chick embryos were injected on embryonic day 2 with plasmid DNA- polymer mixtures. The injections were made into the midbrain vesicle, with the injection pipette directed ventrally. The plasmid DNA encoded the enzyme alkaline phosphatase, allowing transgenic cells to be detected in a histochemical procedure as dark purple cells (See Figure 8 arrows, arrowheads). In the embryo whole mounts shown from a dorsal view, the dorsal midbrain has been cut away to show the labeled cells in ventral midbrain. The polymers E9 and E3 were very efficient transfectants (++/+++, many positive cells) and compared very favorably with SuperFect (+, a few labeled cells). Polymer B 17/2100Da is the only PBAS teratogen tested so far to show transgenesis in vivo (See Figure 8(D) (arrow)).

Figure 8(E) further demonstrates that intravascular delivery of E3 can elicit widespread labeling of the embryonic day 3 chick. Figure 8(G) shows that the TREN polymer T+l is effective at in vivo gene delivery (+++). Figure 8(H) shows a tangential section through rat cortex in which EGFP expression plasmid complexed to Langer reagent B14 elicited limited transfection of cells. The two spots illustrated are 1.5 mm apart and are part of a grid of cortical deposits delivered in this animal. In this experiment, tissue section chartings of the green fluorescence were made and in situ hybridization was carried out for EGFP mRNA to produce a permanent reaction product (See Figure 8(H) arrows).

In summary, of the 49 sulfonamide-containing polymers tested, 18 either killed the embryos or produced embryos with macroscopic deformities (Figure 8(D)). That fact that some of the compounds would be teratogenic was not surprising as such effects were seen with some of the commercial reagents tested (Gene Juice, EMD Bio; TransIT-LTl, Minis Bio; high molecular weight PEI, Sigma). A great advantage of direct in vivo screening is that it allows one to remove toxic compounds from further study. These studies also show that the teratogenic polymers are not effective transgenic agents (Figure 8(D)).

Of the 31 non-teratogenic polymers, 13 produced transgenic cells in embryonic midbrain (scored as +) and another two compounds, E3 and E9, both synthesized using the divinylsulfonamide monomer E, elicited excellent transgenesis (++ to +++; Figure 8(B)₅(C)). Polymer E3 in particular was more effective (level and extent of transgenesis) and more reliable (typically 100% of embryos injected will show transgenesis) than the best lots of SuperFect or the best Langer compound (Langer B 14; Figure 9). It was also found that 4 compounds (C12, Cl 5, E8 and G3) elicited strong and selective labeling of the embryonic lens placode when the polyplex was delivered outside the neural tube to the head surface ectoderm (Figure 8F). In vivo-In vitro Comparisons

To date, all known previous screens for novel cationic polymers started with cell culture tests, with only the most promising in vitro reagents being graduated to in vivo experiments. Alternatively, the approach here is a different one: to devise simple and efficient first-step in vivo screens for efficacy and toxicity.. With this universal approach to in vivo screening, all 31 nonteratogenic polymers were tested in COS-7 cell luciferase transfection assays. Representative findings from this screen are illustrated in Figure 9 and Figure 15. All reagents that showed some in vivo transfection efficacy also had in vitro efficacy. Apart from this finding, however, there were no further relationships between in vitro and in vivo efficacy. Structure-Function Comparisons

The molecular weights and polydispersity index (PDI) of the PBAS polymers were determined by MALDI-TOF-MS (Hanton, 2001) (Table 4). The molecular weight range was 1000-8400 Da. Additionally, some structure-function correlates were identified. First, all PBAS compounds above 5000 Da were either teratogenic or elicited no transfection. Second, compounds that differed in their transgenic profiles fell into characteristic molecular weight ranges: the most effective compounds (++/+++) were within a narrow range of 3900 to 4600 Da, + polymers fell into a lower range (1700-3200 Da) and the polymers eliciting strong and selective lens transgenesis were among the smallest polymers we synthesized (1000-2400 Da). Teratogenic polymers, only one of which elicited any transfection (B 17, see Figure 8(D)), were found across a range of molecular weights (1890- 8400 Da). Additionally, 70% of the teratogens were soluble only in DMSO. An additional feature of the PBAS synthetic reaction is that reaction time controls polymer size. Additionally, an effect of size on polymer properties has also been observed. To illustrate this point two E3 polymers in the ++/+++ range (3900 and 4500 Da) were synthesized. Both showed efficient transfection in vivo. In contrast, polymer E9 (4100 Da), when synthesized as a larger polymer (7900 Da and 8400 Da) was ineffective as a transfectant and became teratogenic. Additionally, the E3 polymers of 3900 and 4500 Da show a dissociation: both are in vivo and in vitro transfectants, but E3/3900 is more effective in vivo whereas E3/4500 is better for cell culture.

An important proposed mechanism for endosomal escape of polyplexes is the "proton sponge" effect, which entails a proton buffering capacity in the polymers. Acid- base titration measurements showed a wide buffer range for most of the polymers. However, in vivo gene transfer efficiencies do not appear to depend on buffering capability. For example, the most effective in vivo polymers presented very different buffering ranges: a narrow 5.1-5.4 range for E3 and a much wider 5.2-7.2 range for E9.

In addition, the size of E9 polyplexes with AFM has been studied. These studies showed spherical complexes of varying diameters ranging from 30 nm to 100 nm. This is well below the 200 nm cutoff reported for polyplex uptake (Rejman et ah, 2004) and interestingly is within the range to be endocytosed through clathrin-independent mechanisms (Conner and Schmid, 2003).

Example 3

As described above, several PBASs were identified as very efficient gene transfer polymers in embryonic chick brain and fare very well in comparison to previously described polymers. The polymers as a group, however, showed diverse biological and structural features, and it is likely that the gene transfer mechanisms, especially the internalization of polyplexes, differ for different polymers. As such, the effects of gene transfer pathways, molecular weight, particle size and surface charge on gene transfer efficiency for the PBAS polymers can be determined. In addition, the efficacy of the polymers in mediating gene transfer after vascular delivery and mature brain injections can be determined.

A great attraction of the PBAS polymers is that they have the flexibility in chemical modification to allow the introduction of bioactive molecules designed to overcome barriers in the gene transfer process. This modifiability also allows for a more systematic structure/function analysis. As such, the gene transfer properties of the synthesized PBAS compounds as well as the ability of directed structural modifications to improve gene delivery can be determined.

Physical and Biological Characterization of First Generation PBAS Polyplexes

For the PBAS polymers, a number of transfectants for chick embryo brain were identified, two of which are very efficient (E3, E9). In addition, the fact that in vitro efficacy is not a reliable indicator of in vivo success was also confirmed. Not all in vivo systems are the same, however, so the polymers can be studied in two other, very different biological delivery systems: transvascular injection in chick embryos and intracranial deposits into post hatchling chick brain. The transvascular screens are of great interest for possible gene therapeutics and are easy to do in the chick embryo. The brain studies are not so simple, but finding polymer systems that efficiently deliver plasmid to postmitotic neurons would be a tremendously valuable neurotechnology and may be of use in solid tissue transgenesis generally, including delivery to brain tumors. Therefore, all non- teratogenic PBAS polymers can be studied for transvascular transgenesis. Because the brain injections assays are more demanding, they can be limited to those polymers that show the best in vivo transfection efficacy in the embryonic midbrain and vascular assays. PBAS polymer transgenesis can also be tested in primary neuronal cultures, and any particularly potent agents in this assay can be studied in brain transfections.

Additionally, a panel of polymer characterizations (described below) can be carried out on the PBAS polymers, as with the polymers described elsewhere herein. Highlights of this characterization include size and charge measurements of the nonteratogenic polymers. As described above, the sizes of the E9 polyplexes were found to be in the 30-100 nni range. This range is at the lower end of what has been reported for polyplex sizes previously, and raises the possibility that the particular efficacy of E9 may be simply due to its size. Knowing the sizes of the other PBAS polyplexes will address this possibility. In addition, an in vitro cell biological study of the dependence of PBAS polymer efficacy on clathrin-dependent and -independent pathways can be performed. Finally, as described elsewhere herein, a test can be performed to determine whether PBAS polymers resynthesized to a higher molecular weight (3900-4500 Da) present greater in vivo efficacy. Sugar-functionalized PBAS polymers

The PBAS polymers described above were synthesized as primitive polymers that contain no special functional moieties for targeted biological interaction. Many of these polymers do, however, possess functional groups that allow further chemical manipulation, an advantageous property for any combinatorial library geared towards screening a large number of compounds. Specifically, polymers containing hydroxyl groups that can be reacted with monosaccharides via the formation of glycosides are of interest. Monosaccharides have been used in functionalizing polycationic polymers, such as polylysine, for improving gene delivery efficiency and targeting special cell types (Hashida et al, 2001 ; Hashida et al, 1998). The resulting polymers can also help to shield the positive charge on DNA nanoparticles, thereby hindering unwanted biological interactions and aggregation. Several of these polymers have been synthesized, examples of which are as follows:

Studies on these polymers show that the biological properties of sugar- functionalized PBAS are dependent on the structures of both monosaccharides and polymers. For example, although polymer E9 was shown to exhibit excellent gene transfer efficiency in vivo, the polymer E-9-g, obtained from modification of E9 with glucose, transfects only the lens in the chicken embryo. Polymer E9 became teratogenic after modification with galactose and mannose. By contrast, polymer Bl 1 is insoluble in aqueous medium, but became very soluble after functionalization with glucose (B-11-g) and exhibited very good gene transfection efficiency in vivo and in vitro. Functionalization of BI l with galactose made the polymer soluble in aqueous solution, but rendered very little efficiency in gene transfection. This system provides an opportunity for systematic variation across a large number of functional polymers because there are 16 hexoses and many PBAS hydroxyl groups. Folate-Functionalized PBAS Polymers

The route of uptake of plasmid DNA complexes strongly affects the efficiency of gene expression (Rejman et al, 2006). In particular, the internalization pathway of caveolae-mediated endocytosis appears to be particularly important for some polyplexes. If this conclusion is correct, then DNA particles with attached ligands that target caveolae (or lipid rafts) may show enhanced gene transfer efficiency. As discussed above, Anderson's group has shown that folate could be internalized efficiently via caveolae (Anderson, 1998; Anderson et ah, 1992; Lee and Huang, 1996). Gene transfer vectors functionalized with folate, both lipidic and polymeric, have been studied for their ability to specifically target tumor cells (Lee and Huang, 1996; Ward et ah, 2002). With this in mind, folate can be incorporated into the PBAS system to assist in targeting caveolae and enhancing gene transfection efficiency. The synthesis of PBAS containing folate can be executed as shown in Figure 10.

Modified PBAS Polymers

The experiments discussed above indicated the importance of binding strength between vector and DNA. Tight binding may lead to difficulty in uncoupling of DNA from vector molecules to permit transcription (Hama et al, 2006). At the same time, the need to deal with multiple gene delivery barriers imposes restrictions on structural variation. For example, the amino groups used as the basic units in previous schemes have only a few discrete forms (1°, 2° or 3° amine and amido amine), which limit our options in selecting a proper cationic unit for effective gene transfer. To address these issues, multifunctional polymer vectors that allow fine-tuning many properties systematically can be prepared (See Figure 11). For example, modified PBAS polymers where the sulfonamide linkage is a side chain and where modifications can be titrated in combinatorially can be prepared.

It is known that the basicity of amino compounds can be affected by indirect substituents in alpha or beta positions via inductive effect. The sulfone is an electron- withdrawing group that reduces the basicity of the amino group. Linear polyethylenimine (PEI) has shown high efficiencies in gene transfer (Boussif et ah, 1995), but PEI cytotoxicity remains a major concern. Systematic modifications to mask some of the nitrogen atoms have been carried out by many groups with mixed results (Forrest et ah, 2004; Pack et ah, 2005). The modified polymers described here start from the linear PEI containing a ketone terminal group, which can be synthesized via a living ring opening polymerization of 2-alkyloxazolines with an initiator containing triflate and ketone, followed by acidic hydrolysis (Figure 11). The vinylsulfonamide-containing PEG group will react with the secondary amine efficiently. The alkylated amines have a reduced basicity, which also broadens the pH buffer range of the resulting polymers and may, at least according to the proton sponge hypothesis, enhance their capacity for endosomal escape. The PEG groups help to protect the polyplexes in blood stream and can be cleaved in cytoplasmic environments through the labile ester bonds. The ketone group can be used to conjugate with recognition ligands via chemo-ligation. In this system, folate can be used as the ligand so that the resulting polymers can be compared with those described above (See Figure 10). The hydrozone linkage formed is labile towards low pH so that the folate can be released after internalization. This polymer system therefore integrates multiple functions and can have greater gene transfer efficiency both in vivo and in vitro than PEI and PBAS.

Example 4

Development of Novel Polycationic Polymers Based on Model Translocases

Electrostatic interactions are responsible for the adhesion of cationic polyplexes to cell surfaces. Experimental results have shown that anionic proteoglycans are involved in endocytosis (Kopatz et al., 2004; Sandgren et al, 2004). As shown by in vivo studies, however, net positive charges on the polyplexes also have a side effect of nonspecific interactions with biological molecules and membranes. Here a new class of polymeric vectors that contain sulfonamides of tris(aminoethyi)amine (TREN) are described. Smith and colleagues discovered that TREN acts as a synthetic translocase that facilitates phospholipid translocation across vesicular and erythrocyte membranes (Boon et al., 2002). Since the sulfone group has a strong electron withdrawing ability, the NH groups on sulfonamide have an increased acidity. The molecular geometry makes this compound capable of forming a tridentate complex with one of the phosphate oxygens via the formation of hydrogen bonds (See Figure 12). In the case of zwitterionic phosphatidylcholine, it is the neutral form of the translocases that rapidly associates with the phosphate portion of the phosphotidylcholine head group. When the pH is reduced, the central amino nitrogen can be protonated, which will produce changes in the chelation mode. As such, TREN can be utilized as the recognition ligand to interact with the head phosphatidylcholine and other zwitterionic phospholipids on the cell surface, by which the positive surface charge on DNA particle can be reduced. Where the phospholipid compositions of eukaryotic cell membranes have been analyzed, they have proved to be strikingly asymmetric. In erythrocyte membranes, for example, most of the sphingomyelin and phosphatidylcholine is localized to the outer leaflet (Zachowski, 1993). These interactions help enhance internalization of the polyplexes. The polyplexes formed with these vectors can also benefit from binding mode change in pH responsive TREN for effective escape from the endosomes. Polysulfonamides Bearing Multiple Chelating TREN The first polymer system that can be studied in detail is shown in Figure 12. It is a copolymer system that has variable parameter x/y to fine tune the polymer composition. This polymer system contains the tetraglycol linkage for enhancing solubility of the resulting polymers in aqueous solution. The Boc-protected triamino co-monomer is used to give the polymers a broader pH response. Several of the copolymers shown in Figure 12 have been synthesized. In vivo tests of gene delivery indicated that polymers with x:y = 4:6, 1:0 and 0:1 have some efficacy as transfection vectors.

In order to assess the effect of TREN, similar polymers with tricarboxylic amides were prepared. It was shown that the corresponding tricarboxylic amides have a much weaker chelating capability. PBAS Containing TREN for Comparison with PBAS

The PBAS system described herein has several polymers that exhibit excellent gene delivery efficiency. A monomer T has been prepared, which can be utilized to prepare PBAS according to the methods described herein. In addition, two polymers have been synthesized from monomer T with monomers 1 and 7 (as the monomers are identified in Figure 13). Polymer T+7 is insoluble in aqueous solution, but polymer T+l produced in vivo gene delivery as successful as the best of the PBAS compounds (+++, Figure 8(D)).

To assess the contribution of TREN to T+l efficacy, a series of copolymers were synthesized with TREN ("T'^? in Figure 13), divinyl sulfonamide E (see Figure 2) and monomer 1 (as noted in Figure 13) in which TREN was progressively replaced with E. This yielded polymers T+l (100% TREN), X2 (80% TREN), X3-X5 (60-20% TREN) and X6 (0% TREN, identical to polymer E6, as enumerated in Figure 2). The results were clear. There was a systematic decrease in in vivo efficacy with decreasing TREN contributions (T+l, +++; X2, ++; X3-X5, +/++; E6, 0/+).

Additionally several analogs of polymer T+l were prepared in which the key structural features are varied systematically (Figure 16). It was found that the polymer with best efficacy in vivo (+++) is polymer T+l . When the three sulfonamides in polymer T+l are replaced with three carbonyl amides (polymer T-ca in Figure 16), the in vivo efficacy is greatly reduced (+ for T-ca). The acidity of the NH in amide is much weaker than that in sulfonamide, as is the hydrogen bonding of the triamides to one of the phosphate oxygens in the phospholipid. This serves as evidence that the incorporation of TREN moieties enhances the gene delivery efficiencies. However, while TREN moieties significantly increase the transfection efficacy in vivo, the imidazole units in polymer T+l are also important. When the imidazole units are replaced with amine free dithiols (T-dt), the efficacy also decreases significantly (+ for T-dt). In addition, the central nitrogen atom in TREN moieties is important. Replacement of the N atom with -CH- moiety (T-cc and T-ct) led to dramatic decrease in gene transfection efficacy. These results indicate the advantages of utilizing the TREN moiety for gene transfer.

Linear PEI with Mono-TREN Moieties

A direct test on the effectiveness of TREN moiety on gene transfer process can also be performed by preparing a well-known polymer with TREN group attached at a well defined position. PEI with terminal TREN groups can be synthesized and tested as set forth in Figure 14. The thiol terminated PEI can be synthesized with a disulfide-terminating group. The thiols react much faster than imine towards the vinylsulfonamide in Michael addition reaction. Consequently, the final polymers can be prepared in this simple procedure.

Example 6

The above described polymers can also be tested for toxicity and transfection efficiency in chicken embryos. Transfection Constructs

Transfection efficiency can be tested in tissue using the pEFX vector, which has been derived from the pEFl/Myc-His C plasmid (Invitrogen) by excising the neomycin cassette. Two constructs have been engineered: pEFX-AP, which drives expression of human placental alkaline phosphatase (Fields-Berry et ah, 1992), and pEFX-tdTomato, which directs production of a dimeric red fluorescent protein, which in our hands is more sensitive than EGFP (Shaner et al, 2005). Following tissue harvest, the alkaline phosphatase reaction product can be demonstrated with a simple tetrazolium reaction after tissue heat treatment to destroy endogenous phosphatase activity. tdTomato expression can be studied with fluorescence microscopy using a Leica MZ FLIII scope. All DNA can be prepared with endotoxin-free kits (Marligen Bioscience) or purified through double-banded cesium chloride purification.

The pEFX plasmids are transient vectors for gene delivery. Other plasmid vectors can also be used to explore the ability of the polymers to deliver vectors with the capability of long term transgene expression, a desirable feature for both experimental and therapeutic gene delivery. Polyplex Formation

Solubility of polymers in biocompatible media is not guaranteed. Therefore, three resuspension media - endotoxin-free water (Sigma), 50 mM Na acetate buffer (pH5) and neat DMSO (HybriMax, Sigma), can be tested. All polymers can be diluted to 6.3 mM as stock solutions and combined with plasmid DNA (2mg/ml in endotoxin-free water) in v/v ratios of 1 :2, 1:10 and 1 :40. Depending the predicted N/P (nitrogen to phosphate) ratios of the polyplexes, other ratios can also be tested. Polyplexes can be incubated at room temperature for thirty minutes before delivery and then used within three hours of preparation. Chick Midbrain Transfection Experiments

The transfection capabilities of each polyplex ratio in embryos can also be tested. Positive control embryos transfected with E9 polyplexes can be used in the tests. To carry out these tests, fertilized chicken eggs can be incubated to developmental stage 8-15 (Hamburger and Hamilton, 1951). Eggs can then be windowed for access to the embryo. 50-800nl of polyplex DNA mixed with 0.02% Fast Green can then be injected into the midbrain vesicle through a glass capillary needle introduced through a pinhole opening made in the tectum. The eggs can then be sealed with tape and returned to the incubator. Three days later, the embryos can be harvested and submerged in a 4% paraformaldehyde phosphate-buffered saline solution. After fixation, the embryos can then be processed as wholemounts for phosphatase histochemistry or fluorescence microscopy. The brains can then be scored for levels of transfection (0, nothing; +, 1-10 cells; ++, moderate transfection; +++, excellent transfection).

Example 7

Physical description of the compounds is not only necessary in their synthesis, but also crucial for any property/function correlations as our studies advance. Analyses for this work can include polymer molecular weight profiles and measurements of polyplex size and charge. Initial Physical Characterization

A number of structural characterizations of the above described polymers can be performed. NMR spectrometers and other spectrometers, such as FTIR, can be used to identify the structures of the materials. MALDI mass spectrometer and gel permeation chromatography (GPC) can also be used to determine the molecular weights and their distribution.

Size and Charge

Laser light scattering can be employed to determine the size of the polyplex particles. Zeta potential measurements for studying polyplex charge can be used in assessing transvascular efficacy as well as the success of the PEGylation. Zeta potential can be measured on a Brookhaven ZetaPlus instrument.

Example 8

As discussed above, some PBAS compounds were effective cell culture transfectants, in several examples exceeding the efficacy of the Langer polymer B14. In vitro capabilities both in cell culture and in primary neuronal cultures can be tested for the polymers. Cell culture methods can also be employed to discover what endocytic pathways the various polyplexes employ. Cell Culture

COS-7 cells can be grown in 96-well plates in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and antibiotic. For transfections, the medium can be replaced with OPTI-MEM (Invitrogen) supplemented with HEPES. Polyplex solutions can then be added to the cells and incubated for twelve hours. The media can then be changed to a phenol red-free growth medium and incubated for two days before harvesting. Positive control transfectants for the cell culture experiments can include PEI (Sigma) and Trans-IT LTl (Minis Bio). Transfection efficiency can subsequently be measured using the firefly luciferase reporter plasmid pRSV-Luc. Luciferase activity can then be detected with the Tropix luciferase assay kit (Applied Biosystems) and quantified with a bioluminescence plate reader. Primary Neuronal Culture

Polymer efficacy can also be tested in transfection experiments on embryonic day 6 chick telencephalon tissue culture generated by mechanical dissection and plating on polylysine-coated 24-well plates. These cultures are enriched to over 90% with neurons (Domowicz et al, 2003). Transfection efficiency can then be assessed with luciferase assays for transgene expression levels and with alkaline phosphatase staining for counts of percent neurons transfected. Trans-IT (Minis Bio) can serve as the positive control transfectant. Cell Biology Whether the above described transgenic polyplexes can be internalized by clathrin- dependent mechanisms or by clathrin-independent mechanisms including caveolae or lipid rafts can also be tested (Conner and Schmid, 2003; Rejman et al, 2006). These studies can be carried out on HeLa cells transfected with luciferase reporter plasmids. The cells can be treated with chlorpromazine (10 micrograms/ml) to perturb clathrin metabolism. The dependency on caveolae uptake can be investigated with the specific blockers fϊlipin (5 micrograms/ml) and genistein (200 microM). Other manipulations such as cholesterol depletion with methyl-beta-cyclodextrin and lovastatin and disruptions of endosome maturation with nocodazole can also be tested. Each of these drugs are available from Sigma.

Example 9

Initial screening of in vivo efficacy can be performed by neural tube injection into the CSF with spread to cells lining the ventricular surface of the midbrain. A secondary screen of effective in vivo transfectants can involve two additional tests: intravenous delivery and solid tissue injections into brain.

Intravenous delivery is an attractive system for targeting cells in heavily vascularized tissues, including tumors. Current testing methods involve injections of tens to hundreds of micrograms of DNA into mouse tail veins (Goula et al, 1998; Zou et al, 2000). As exemplified above, a much easier screen for transvascular efficacy has been established in the chick embryo. Polyplexes are delivered to the right vitelline artery, where they travel first to the developing yolk sac and then return to the embryo. Embryos are harvested after eight hours and stained for transgene expression. As described above, compound E9 has been found to transfect cells throughout the embryo (Figure 8E). Accordingly E9 can be used as a positive control in these experiments.

An ability of polymer complexed DNA to transfect postmitotic neurons efficiently in vivo would be a neurotechnology of great benefit to the scientific community and can have applications to human therapeutics. As such, polyplex efficacy can be tested by making deposits into the dorsal telencephalon of hatchling chickens. For these experiments, the Langer compound B 14 can be used as a positive control in these experiments, which has some transfection efficiency in adult rat brain injections (although this may be restricted to glial cells proliferating at the injection site; Figure 8H). Transvascular Delivery Fertilized chicken eggs can be incubated to embryonic day 3 (st 19) and windowed. ρEFX-AP-containing polyplexes can be delivered in a 1 -microliter bolus through a glass micropipette into the vitelline artery. At least six embryos can be used for each ratio tested. Injected embryos can then be incubated for eight hours and harvested for phosphatase histochemistry. Brain Surgeries

White Leghorn chicks up to 400 g in weight and three weeks of age can be anesthetized by subcutaneous injection of ketamine (100 mg/kg) and xylazine (20mg/kg). Supplemental doses can also be given as needed. Observing aseptic procedures, the animals can be placed in a Kopf stereotaxic instrument with small bird adaptor, the scalp can be incised and a small hole can be made with a dental drill in the calvarium, which has ossified by 2-3 weeks of age. Polyplex deposits can be delivered into the dorsal surface of the telencephalon through a ImI Hamilton syringe attached to a stereotaxic holder and controlled by a picospritzer pressure system (WPI). Each telencephalon can receive a series of injections of different polyplex ratios and volumes, the deposits arranged as if on a grid to facilitate histological identification. Two days following surgery, the animal can be given a lethal dose of pentobarbitone (lOOmg/kg) by intraperitoneal injection. Once deeply anesthetized, the animal can be perfused transcardially with fixative solutions. The brain tissue can then be cut in tangential sections, mounted on slides and processed for alkaline phosphatase histology to detect transfected neurons.

Example 10

Low toxicity, which is essential for any in vivo polyplex, can be initially assessed by lethality and teratogenicity in the chick embryonic brain experiments. For any experimental polymers that pass the initial screen, tissue toxicity can be studied in three ways. First, in situ hybridization of midbrain wholemounts can be used to see whether polymer delivery disrupts endogenous gene expression or the normal tempo of embryonic development. Such tests have been previously used to show that, for experimenters with embryological finesse, in ovo electroporation of marker genes into embryonic ventral midbrain does not distort its normal development (see Agarwala et ah, 2001). Second, upregulation of gene expression sentinels of tissue stress can be evaluated at sites of plasmid delivery. For these experiments, the expression of an unfolded protein response gene, Stanniocalcin-2/STC2, which is upregulated at the transcriptional level in response to cellular stress (Ito et ah, 2004) can be used, and the interferon response gene EIF2AK2/PKR (Samuel, 1991). Because electroporation of plasmid alone can elicit an interferon response (Akusjarvi et al, 1987; Chesnutt and Niswander, 2004), care should be taken in these tests to test polymer alone as well as polymer complexed with DNA. Third, frank cell death can be tested at and near polymer injection sites with in situ end labeling (ISEL) methods. In Situ Hybridization

Two-color wholemount and section in situ hybridization methods can also be utilized (Agarwala et al, 2005; Grove et al, 1998). To test whether polymer delivery disrupts normal gene expression, a large battery of cDNAs that mark patterned cell types in chicken embryonic ventral midbrain can be used (Sanders et al. , 2002). Polymer-induced tissue stress can be probed using cDNAs for chicken EIF2AK2 and STC2 that have been previously isolated by PCR employing primers derived from chicken EST sequences (Boardman et al, 2002). Cell Death Monitoring

Cell death can be tracked by a modification of the ISEL procedure (Miller and Ragsdale, 2000; Yamamoto and Henderson, 1999). The key feature of this method is that digested DNA ends are labeled with DIG-dUTP in a terminal transferase reaction, and are detected as in an in situ hybridization experiment. For polymer toxicity experiments in which marker plasmids such as AP or tdTomato are introduced, the two-color in situ method can be modified (Grove et al, 1998) to look for increased apoptosis at sites of transfection by combining ISEL with hybridization histochemistry for AP or tdTomato mRNA. Brain Slice Injections

Telencephala from chick embryos at embryonic day 12 (a time at which the tissue consists mostly of postmitotic neurons and glia) can be harvested and sliced at 200-400 microns on a Mcllwain tissue chopper. The slices can then be placed on a Millipore filter in a drop of Neurobasal medium containing 20 mM glucose, antibiotics and 10% fetal bovine serum, and incubated in a 6% CO₂ incubator at 37°C for 1-2 days. Each slice can then be injected with multiple deposits of polyplex arranged as a grid on the tissue to facilitate analysis. The injections can be 200 nl in volume delivered through a glass micropipette attached to a Prior micromanipulator and controlled by a picospritzer. Slices can then be incubated for a further 2 days before fixation.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

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Claims

CLAIMSWhat is claimed is:

1. A composition, comprising: a sulfonamide-containing polymer and one or more biomolecules wherein the sulfonamide-containing polymer comprises Formula I:

— \— [multi-sulfonamide] — [linker]—) — n Formula I wherein the multi-sulfonamide component is a residue of a multi-sulfonamide monomer that contains two or more sulfonamide moieties, and the linker component is a residue of a linker monomer and links one multi-sulfonamide residue to another, and n is from 2 to 10,000.

2. The composition of claim 1, wherein the biomolecule is a nucleic acid.

3. The composition of any of claims 1 -2, wherein the biomolecule is DNA.

4. The composition of any of claims 1 -3 , wherein the sulfonamide-containing polymer has a molecular weight of from about 500 Da to about 1,000,000 Da.

5. The composition of any of claims 1-4, wherein the sulfonamide-containing polymer has a molecular weight of from about 1,000 Da to about 8,500 Da.

6. The composition of any of claims 1-5, wherein the sulfonamide-containing polymer has a molecular weight of less than 5,000 Da.

7. The composition of any of claims 1-6, wherein the composition is from about 1 to about 1000 nm in diameter.

8. The composition of any of claims 1 -7, wherein the sulfonamide-containing polymer is functionalized with a targeting moiety.

9. The composition of any of claims 1-8, wherein the sulfonamide-containing polymer is functionalized with a saccharide.

10. The composition of any of claims 1 -9, wherein the sulfonamide-containing polymer is functionalized with folate.

11. The composition of any of claims 1-10, wherein the linker monomer comprises nucleophilic functional groups and the multi-sulfonamide monomer comprises electrophilic functional groups.

12. The composition of any of claims 1-11, wherein the linker monomer comprises electrophilic functional groups and the multi-sulfonamide monomer comprises nucleophilic functional groups.

13. The composition of any of claims 1-12, wherein the linker monomer and the multi- sulfonamide monomer both comprise nucleophilic and electrophilic functional groups.

14. The composition of any of claims 1-13, wherein the multi-sulfonamide residue and linker residue are linked by an amine, imine, amide, ester, thioester, ether, sulfides, disulfides, carbonate, urethane, urea, thiourea, or sulfonamide bond.

15. The composition of any of claims 1-14, wherein the multi-sulfonamide residue contains two sulfonamide moieties.

16. The composition of any of claims 1-15, wherein multi-sulfonamide residue contains three, four, five, six, seven, eight, nine, ten, or more sulfonamide residues.

17. The composition of any of claims 1-16, wherein multi-sulfonamide monomer comprises Formula II:

R¹ R²

O /xO O /^•%■O Formula II wherein Z¹ and Z² are the same or different and comprise one or more nucleophilic or electrophilic functional groups;

R¹ and R² are, independent of one another, hydrogen, substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol; and X is a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol.

18. The composition of claim 17, wherein either Z¹ or Z² or both comprise an amine, a cyclic amine, a polyamine, an amide, a polyamide, a polyether amine, or a dendrimeric polyamine.

19. The composition of any of claims 17-18, wherein either Z¹ or Z² or both are — NH₂, — NHR¹⁰, or — NR¹⁰2, wherein each R¹⁰ is, independent of the others, a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol.

20. The composition of any of claims 17-19, wherein either Z¹ or Z² or both are a primary alcohol, a secondary alcohol, a tertiary alcohol, a dedrimeric alcohol, or a polymeric alcohol.

21. The composition of any of claims 17-20, wherein either Z¹ or Z² or both are a primary thiol, a secondary thiol, a tertiary thiol, a dendrimeric thiol, or a polymeric thiol.

22. The composition of any of claims 17-21 , wherein either Z¹ or Z² or both are an aldehyde, an acid, an acid halide, an ester, an activated ester, an acid anhydride, a mixed anhydride, an imine, a sulfonyl chloride, a halide, or a ketone.

23. The composition of any of claims 17-22, wherein either Z¹ or Z² or both are an isocyanate or isothiocyanate.

24. The composition of any of claims 17-23, wherein either Z¹ or Z² or both are an alkenyl or alkynyl group adjacent to the sulfonamide moiety.

25. The composition of any of claims 17-24, wherein X is a substituted or unsubstituted Ci-C₆ branched or straight-chain alkyl, a polyalkylene, a Ci-C₆ branched or straight- chain alkoxy, or a C₂-Cβ branched or straight-chain alkyl where one or more of the carbon atoms are substituted with oxygen, sulfur, or nitrogen.

26. The composition of any of claims 17-25, wherein X is -CH₂CH₂NR²¹CH₂CH₂-, where R²¹ is -CH₂CH₂NHSO₂-p-CH₃-Ph.

27. The composition of any of claims 17-26, wherein X is -CH₂CH₂CHR²¹CH₂CH₂-, where R²¹ is -CH₂CH₂NHSO₂-p-CH₃-Ph.

28. The composition of any of claims 17-27, wherein Z¹ and Z² are electrophilic moieties and X is a substituted or unsubstituted alkyl group.

29. The composition of any of claims 17-28, wherein the multi-sulfonamide monomer comprises Formula H-I:

wherein each R⁶ is, independent of the others, hydrogen, substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol; and m is from 1 to 25.

30. The composition of any of claims 17-29, wherein Z¹ and Z² are electrophilic moieties and X is a substituted or unsubstituted cycloalkyl or cycloheteroalkyl group.

31. The composition of any of claims 17-30, wherein the multi-sulfonamide monomer comprises any one of Formulae II-II through H-X:

wherein Q is NR⁶, O, S, or CH₂; each R⁶ is, independent of the others, hydrogen, substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol; and

32. The composition of any of claims 17-31 , wherein Z¹ and Z² are electrophilic moieties and X is an ether or polyether.

33. The composition of any of claims 17-32, wherein the multi-sulfonamide monomer comprises Formula H-XI:

Formula II-XI wherein each R⁰ is, independent of the others, hydrogen, substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol; and m is from 1 to 25.

34. The composition of any of claims 17-33, wherein the multi-sulfonamide monomer comprises any one of Formula A through G:

35. The composition of any of claims 1 -34, wherein the multi-sulfonamide monomer comprises:

36. The composition of any of claims 1-35, wherein the linker residue is from 1 to 25 atoms in length.

37. The composition of any of claims 1 -36, wherein the linker residue comprises a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol.

38. The composition of any of claims 1-37, wherein the linker residue comprises a substituted or unsubstituted Ci-C₆ branched or straight-chain alkyl, a polyalkylene, cycloheteroalkyl, a Ci-C₆ branched or straight-chain alkoxy, or a C₂-C₆ branched or straight-chain alkyl where one or more of the carbon atoms are substituted with oxygen, sulfur, or nitrogen.

39. The composition of any of claims 1-38, wherein the linker monomer comprises an amine, a cyclic amine, a polyamine, an amide, a polyamide, a polyether amine, or a dendrimeric polyamine.

40. The composition of any of claims 1 -39, wherein the linker monomer comprises — NH₂, — NHR²⁰, or — NR²⁰ ₂, wherein each R²⁰ is, independent of the others, a substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol.

41. The composition of any of claims 1 -40, wherein the linker monomer comprises a primary alcohol, a secondary alcohol, a tertiary alcohol, a dedrimeric alcohol, or a polymeric alcohol.

42. The composition of any of claims 1-41, wherein the linker monomer comprises a primary thiol, a secondary thiol, a tertiary thiol, a dendrimeric thiol, or a polymeric thiol.

43. The composition of any of claims 1-42, wherein the linker monomer comprises an aldehyde, an acid, an acid halide, an ester, an activated ester, an acid anhydride, a mixed anhydride, an imine, a sulfonyl chloride, a halide, or a ketone.

44. The composition of any of claims 1-43, wherein the linker monomer comprises an isocyanate or isothiocyanate.

45. The composition of any of claims 1 -44, wherein the linker monomer comprises any one of Formulae III-I through III-X:

HtVII iπ-viπ

wherein Q is NR⁶, O, S, or CH₂; each W is, independent of the others, N or CH; each R⁶ is, independent of the others, hydrogen, substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, alkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, cycloalkynyl, cycloheteroalkynyl, aryl, heteroaryl, carboxylate, carbonate, ether, polyether, ester, polyester, amine, polyamine, polyalkylene, silyl, dendrimeric polyamine, or dendrimeric polyol; and m is from 1 to 25.

46. The composition of any of claims 1 -45, wherein the linker monomer comprises any one of Formulae 1 through 24:

47. The composition of any of claims 1 -46, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

48. The composition of any of claims 1-47, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

49. The composition of any of claims 1-48, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

50. The composition of any of claims 1-49, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

51. The composition of any of claims 1-50, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

52. The composition of any of claims 1-51, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

53. The composition of any of claims 1-52, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

54. The composition of any of claims 1-53, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

55. The composition of any of claims 1-54, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

56. The composition of any of claims 1-55, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

57. The composition of any of claims 1-56, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

58. The composition of any of claims 1-57, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

59. The composition of any of claims 1 -58, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

60. The composition of any of claims 1 -59, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

61. The composition of any of claims 1 -60, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

62. The composition of any of claims 1-61, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

63. The composition of any of claims 1-62, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

64. The composition of any of claims 1-63, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

65. The composition of any of claims 1-64, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

66. The composition of any of claims 1-65, wherein the sulfonamide containing polymer comprises:

wherein n is from 2 to 10,000.

67. A pharmaceutical composition comprising the composition of any of claims 1-66 and a pharmaceutical carrier.

68. A method of preparing a sulfonamide-containing polymer, comprising: reacting a multi-sulfonamide monomer that contains an alkenyl or alkynyl adjacent to a sulfonamide moiety, with a linker monomer comprising an amine functional group in the presence of a Lewis acid.

69. The method of claim 68, wherein the Lewis acid is LiClO_4.

70. A method of introducing a biomolecule to a subject comprising administering the composition of any of claims 1-66 to the subject.

71. A method of bringing into contact a biomolecule and a cell comprising contacting the cell with the composition of any of claims 1-66, thereby bringing into contact the biomolecule and the cell.

72. A method of modulating a molecule of interest in a cell comprising contacting a cell with the composition of any of claims 1-66 under conditions that allow the biomolecule of the composition to modulate the molecule of interest, wherein the molecule of interest is modulated.

73. The method of claim 72, wherein the cell is in a subject.

74. The method of claim 72, wherein the molecule of interest is a nucleic acid or protein.

75. The method of claim 74, wherein the expression of the molecule of interest is reduced.

76. The method of claim 74, wherein the expression of the molecule of interest is increased.

77. The method of claim 74, wherein the expression of the molecule of interest is enhanced.

78. The method of claim 74, wherein the expression of the molecule of interest is restored.

79. A method of modulating the expression of a biomolecule of interest in a subject comprising administering the composition of any of claims 1-66 to the subject under conditions that allow the biomolecule of the composition to modulate the expression of the biomolecule of interest, wherein the expression of a biomolecule of interest is modulated.

80. A method of treating a subject comprising administering a therapeutically effective amount of the composition of any of claims 1-66 to the subject in need thereof.

81. A method of screening for a biomolecule that modulates a molecule of interest in a cell, comprising a. introducing to a cell comprising a molecule of interest the composition of any of claims 1-66, wherein the composition comprises the biomolecule, b. determining the activity of the molecule of interest compared to a control, wherein a change in activity of the molecule of interest indicates a biomolecule that modulates the molecule of interest.

82. A method of screening for the effect of a biomolecule in a cell comprising a. introducing a biomolecule to a cell comprising administering the composition of any of claims 1-66, b. determining the effect of the biomolecule compared to a control, wherein a change from the control indicates the effect of the biomolecule.

83. A kit comprising any one or more of the compositions of claims 1-66.