EP1446008A2 - Compositions and methods for controlled release - Google Patents

Abstract

Methods and compositions are provided for the controlled release of an active agent. The composition includes an oligonucleotide scaffold and a substantially complementary oligonucleotide factor. An active agent is attached to the oligonucleotide factor. The two oligonucleotides are differentially degradable, so that degradation of the scaffold occurs a faster rate than the degradation of the factor. As the scaffold degrades, the factor is released. The active agent can be a therapeutic agent, or a diagnostic agent.

Description

Compositions and Methods for Controlled Release

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of provisional application Serial No. 60/336,344, filed October 18, 2001, the contents of which are incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

Not applicable

BACKGROUND OF THE INVENTION

[0002] As the field of molecular biology has advanced, the uses of oligonucleotides have proliferated to include polymerase chain reaction (PCR) applications, genomic microarrays, biosensors and molecular computing in the in vitro setting, and gene-targeted therapies such as antisense and ribozyme treatments in the in vivo setting. These applications have stimulated a significant amount of research aimed at characterizing the chemistry of oligonucleotides as well as oligonucleotide modifications. For example, the advent of genomic microarrays, or "gene chips," has led to the development of oligonucleotides capped with functional groups that can be easily attached to and patterned on a solid surface. The investigation of oligonucleotide-based antisense and ribozyme therapies in vivo has led to the study of different DNA-backbone modifications, such as the phosphorothioate backbone, that can prolong the oligonucleotide' s half-life in serum or other nuclease-containing body fluids. [0003] Recently, attention has focused not only on modifying the chemical nature of oligonucleotides but also in finding improved ways to release them in desired locations. [0004] There is great interest in developing methods and compositions for the controlled release of oligonucleotides for therapeutic purposes. In the case of improving the in vivo half-life of antisense oligonucleotides, for example, controlled release strategies employing synthetic polymers that can bind and protect DNA, and then release it gradually once in vivo, appear promising. In these methods, the controlled release of DNA employ synthetic polymeric scaffolds that form an ionic bond with the DNA, the cationic polymer binding to the anionic DNA. Such chemicals include poly-lysine (see Leonetti et al., "Antiviral activity of conjugates between poly(L-lysine) and synthetic oligodeoxyribonucleotides," Gene, 72(l-2):323-32, 1988, and Sakharov et al., "Polylysine as a vehicle for extracellular matrix- targeted local drug delivery, providing high accumulation and long-term retention within the vascular wall," Arterioscler. Thromb. Vase. Biol. 21(6):943-8, 2001) and poly-arginine (see, for example PCT publication WO 98/52614). Sustained release of the DNA occurs when the polymer-DNA complex is placed in solution and gradual hydrolysis of bonds in the polymeric scaffold causes polymer breakdown. The ionically-bound DNA is slowly released into solution where it can then exert the intended effect. Problems with such systems mainly stem from the nature of the polymers used. In particular, the breakdown products generated by hydrolysis often cause major chemical changes in the immediate vicinity. Often undesirable, these results can include the perturbation of pH levels, exertion of toxic effects on cells, elicitation of immune responses in vivo, and the like. [0005] In addition to therapeutic applications, there is also a strong interest in developing methods and compositions for the controlled release of oligonucleotides for diagnostic and assay purposes.

[0006] In view of the foregoing, there remains a need in the art for more elegant platforms for controlled oligonucleotide release. Ideally, the scaffold, or carrier material, employed in a controlled release platform for the oligonucleotide should have several characteristics. First, prior to the desired time or condition of release, the scaffold should be able to bind the oligonucleotide in a way that does not destabilize or degrade it. Preferably, the scaffold should actually serve to protect or stabilize the oligonucleotide. Second, at the desired time or condition of release, the scaffold should be able to reliably release, or unbind, the oligonucleotide. Optimally, this should happen at a predictable rate with sustained release over a usable time period, such as minutes to days. Third, the scaffold itself, both before and after release, should not cause a major perturbation in the system which could undermine the intended application. For example, it may be desirable to avoid large pH changes, toxicity to cells, and the like. In the case of in vivo applications, biocompatibility of the scaffold material and its degradation products is of utmost importance. Finally, issues of cost, production, assembly (i.e., binding the oligonucleotide to the scaffold), and adaptability must be considered.

[0007] Surprisingly, the present invention provides such compositions and methods for the controlled release of oligonucleotides. SUMMARY OF THE INVENTION

[0008] The present invention includes compositions, methods, and kits for the controlled release of oligonucleotides. In particular, the present invention is based on the discovery that oligonucleotides themselves provide a convenient and efficient platform for the release of oligonucleotides.

[0009] In a first aspect, the present invention provides a method and composition for the controlled release of an active agent. The composition includes an oligonucleotide scaffold and a substantially complementary oligonucleotide factor. An active agent is attached to the oligonucleotide factor. The two oligonucleotides are differentially degradable, so that degradation of the scaffold occurs a faster rate than the degradation of the factor. As the scaffold degrades, the factor is released. The active agent can be a therapeutic agent, or a diagnostic agent. [0010] In a second aspect, the present invention provides a method and composition for the controlled release of an active agent. The composition includes an oligonucleotide scaffold and a substantially complementary oligonucleotide factor. The two oligonucleotides are differentially degradable, so that degradation of the scaffold occurs a faster rate than the degradation of the factor. As the scaffold degrades, the factor is released. Here, the active agent is the oligonucleotide factor itself. [0011] In a third aspect, the present invention provides a method and composition for the controlled release of an active agent from a solid or semisolid support. The composition includes an oligonucleotide scaffold and a substantially complementary oligonucleotide factor. The scaffold is attached with a solid support. An active agent is attached to the oligonucleotide factor. The two oligonucleotides are differentially degradable, so that degradation of the scaffold occurs a faster rate than the degradation of the factor. As the scaffold degrades, the factor is released from the support.

[0012] In a fourth aspect, the present invention provides a kit for the preparation of a controlled release composition. The kit includes a first container holding an oligonucleotide scaffold, and a second container holding a substantially complementary oligonucleotide factor. An active agent is attached to the oligonucleotide factor. The two oligonucleotides are differentially degradable, so that degradation of the scaffold occurs a faster rate than the degradation of the factor. In use, as the scaffold degrades, the factor is released. [0013] The advantages of this approach are severalfold. The scaffold and its nucleotide breakdown products exhibit low toxicity and high biocompatibility. The scaffold provides a predictable and biologically relevant release mechanism, such as a nuclease. The scaffold- factor complex is amenable to self-assembly. The complex provides stabilization of the oligonucleotide factor by hybridization to the complementary scaffold strand prior to release. Oligonucleotide scaffolds offer flexibility and a well-studied chemistry. Moreover, these methods and compositions can be adapted for both in vitro and in vivo applications and for release by both exonuclease and endonuclease mechanisms.

[0014] Other objects and advantages will become apparent from the following detailed description taken in conjunction with the drawings in which:

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 shows a scaffold-factor complex of the present invention.

[0016] FIG. 2 illustrates a partially degraded scaffold-factor complex of the present invention.

[0017] FIG. 3 shows a scaffold-factor complex of the present invention, including a targeting moiety and a therapeutic agent.

[0018] FIG. 4 exemplifies a scaffold-factor complex of the present invention, attached to a surface.

[0019] FIG. 5 shows the results of a gel electrophoresis experiment.

[0020] FIG. 6 illustrates a synthetic scheme. Stainless steel surfaces were derivatized with allylamine to give free amines covalently attached to the metal surface (left). Free amines were then activated and reacted with phosphates on single stranded (degradable) oligonucleotides to give a degradable DNA strand attached to the metal surface (right).

[0021] FIG. 7 shows surface fluorescence after exonuclease III treatment.

[0022] FIG. 8 shows surface fluorescence after serum treatment. [0023] FIG. 9 shows FTIR absorbance on surface after exonuclease III treatment.

[0024] FIG. 10 shows FTIR absorbance on surface after serum treatment.

DETAILED DESCRIPTION OF THE INVENTION [0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

General

[0026] The present invention provides methods and compositions for the controlled release of oligonucleotides, and involves the use of a hybridized complex. The complex includes an oligonucleotide scaffold, and an oligonucleotide factor. The scaffold and factor oligonucleotides are differentially degradable, to provide for the controlled release of the oligonucleotide factor. [0027] For example, the two oligonucleotides may be designed to behave differently in the presence of a nuclease. The oligonucleotide factor is more nuclease resistant, whereas the oligonucleotide scaffold is less nuclease resistant. Thus, the scaffold degrades at a faster rate than does the factor. Typically, an active agent will be attached to the oligonucleotide factor. The active agent can be a therapeutic agent or a diagnostic agent, for example. [0028] In a related example, the present invention provides for a single-stranded DNA molecule ("strand A") for use as a controlled release scaffold for its complementary strand ("strand B") in the presence of a nuclease. Strand A is more nuclease-susceptible than strand B. Prior to release, strands A and B self-assemble to form a double-stranded DNA scaffold- factor complex. Under release conditions, such as the presence of nuclease, strand A degrades at a faster rate than strand B degrades. In time, strand B dissociates from the complex. Because nuclease-degradation is a probability-driven event, not every molecule of strand A will be degraded at exactly the same rate, thus dissociation of strand B in the mixture will occur over time, leading to sustained release. The degradation products of strand A are the monomeric nucleotides, and are non-toxic and biocompatible. In the further descriptions below, strand B is taken to be an oligonucleotide, as these short-length single- stranded DNA molecules have multiple important uses in molecular biology.

[0029] In using an oligonucleotide, instead of a synthetic polymer, as our degradable scaffold, an old molecule (e.g., DNA) is effectively taught the new trick of controlled release. Nucleic acid-based molecules in general have not been considered as a potential material for drug or agent delivery. In addition to the new use of oligonucleotides as a scaffold material, the mechanism of release has also not been described previously. The scaffolds described herein allow the degradable oligonucleotide (strand A) to decompose only when nuclease activity is present. The nuclease can be an exonuclease that degrades DNA from the end of a strand, an endonuclease that degrades DNA intra-strand, or a nuclease having both endonuclease and exonuclease activity, so long as the oligonucleotide to be released (strand B) is more resistant to that type of nuclease. This can be readily accomplished. [0030] The advantages of nuclease-triggered release are several. For in vitro applications, the triggered release allows the onset of degradation and release to be easily controlled; release does not start until nuclease is added to the solution. Prior to the nuclease addition the oligonucleotide scaffold remains stable in solution, unlike polymeric scaffolds. For in vivo use, nuclease-triggered release is particularly convenient because most body fluids, including serum, naturally contain exonucleases and hence can trigger the release process intrinsically. [0031] In addition, many abnormal in vivo conditions, such as inflammation and cell death, lead to the release of endonucleases: enzymes that can degrade DNA strands from the middle rather than from the ends of the strands. The presence of such enzymes can allow the controlled release of oligonucleotide factors, via an endonuclease mechanism, specific to such abnormal environments. An endonuclease mechanism of release can easily be achieved by designing the oligonucleotide factor, via DNA backbone modifications and the like, to be more endonuclease resistant. As a result, the factor degrades more slowly than its substantially complementary oligonucleotide scaffold.

[0032] The use of DNA as a controlled release scaffold for oligonucleotides also presents many advantages. First, unlike synthetic polymers, DNA breaks down into monomeric nucleotides, which are the natural building blocks of DNA. Consequently, issues of biocompatibility and toxicity are not problems. Second, the double-stranded DNA scaffold-oligonucleotide complex is inherently even more stable than single-stranded DNA. Thus, the degradable DNA strand (strand A), prior to release, actually serves to stabilize and protect the bound oligonucleotide (strand B). Third, because the two DNA strands are complementary, the scaffold-oligonucleotide complex self-assembles by hybridization to each other under appropriate therrnodynamic conditions, which greatly simplifies preparation. Additionally, the assembly interactions are largely due to hydrogen bonding, rather than the ionic interactions present with synthetic polymeric scaffolds. This type of interaction and chemistry is well-studied and a variety of modifications can be made to either the scaffold or the released factor optimize the complex for a specific function. Thus, by using DNA as a scaffold for controlled release, the compositions described herein are extremely flexible in terms of design for a particular use.

[0033] For example, by making the same chemical modifications that allow DNA to be attached to the surface of a gene chip, we can have the scaffold DNA strand (strand A) mediate release of a substantially complementary oligonucleotide from a solid surface. This has implications in biosensor technology or for delivering antisense and other therapies off of implanted devices, coronary stents, or grafts. Another useful modification involves the attachment of an antibody or peptide to one end of strand A to confer cell-type specificity, such that the scaffold-oligonucleotide complex can bind the surface of certain cells. The oligonucleotide factor is thereby preferentially taken up by these cells after release. Thus, the unique characteristics of DNA-mediated controlled release clearly offer significant advantages over the polymeric platforms currently used for oligonucleotides. [0034] FIG. 1 through FIG. 4 provide illustrations of the general concept of the present invention. In FIG. 1 a scaffold-factor complex is illustrated in which the scaffold and factor oligonucleotide strands are substantially complementary (prior to a degradation process). FIG. 2 illustrates the degradation process wherein the scaffold oligonucleotide is being degraded while the factor oligonucleotide is resistant to degradation. Cleavage sites are shown in which the degradation products can be designed for particular sizes. A therapeutic agent is also shown which is associated with the factor oligonucleotide. The association can be via either a non-covalent interaction (e.g., ionic association) or via a covalent attachment (not shown). FIG. 3 illustrates another embodiment of the invention in which the scaffold oligonucleotide has an associated targeting moiety at the 3'-end, while the factor oligonucleotide has an associated therapeutic agent at its 3'-end. Finally, FIG. 4 illustrates the release of a factor oligonucleotide from a solid support following degradation of a scaffold oligonucleotide that is attached to the support.

Description of the Embodiments

Controlled Release Compositions [0035] In a preferred embodiment, the present invention provides a composition for the controlled release of an active agent. The composition includes an oligonucleotide scaffold and a substantially complementary oligonucleotide factor. An active agent is attached to the oligonucleotide factor. The two oligonucleotides are differentially degradable, so that degradation of the scaffold occurs a faster rate than the degradation of the factor. As the scaffold degrades, the factor is released. The active agent can be a therapeutic agent, or a diagnostic agent.

Oligonucleotides [0036] As used herein, the term "oligonucleotide" refers to a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. It will be understood by one of skill in the art that oligonucleotide scaffolds can bind factor sequences, however, that lack complete complementarity with the scaffold, depending upon the stringency of the hybridization conditions.

[0037] In a preferred embodiment, the oligonucleotide may include natural bases, such as adenine, guanine, cytosine, thymine, or uracil. Optionally, the oligonucleotide may include modified bases, such as 7-deazaguanosine, inosine, 4-acetylcytidine, dihydrouridine, queuosine, wybutosine, and the like,.

[0038] In a preferred embodiment, the bases of the oligonucleotides are joined by a phosphodiester linkage. In another preferred embodiment, the bases in an oligonucleotide can be joined by a linkage other than a phosphodiester linkage, so long as it does not interfere with hybridization. Thus, for example, oligonucleotides can be peptide nucleic acids (PNAs) in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. Linkages may be chosen to exhibit varying levels of pH sensitivity, or susceptibility to hydrolysis, depending on the needs of the treatment or assay. [0039] In yet another preferred embodiment, either of the oligonucleotides can contain nucleotides which are deoxyribonucleotides or ribonucleotides, as well as known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides and peptide-nucleic acids (PNAs). In a particularly preferred embodiment, the scaffold oligonucleotide is prepared from naturally-occurring nucleotides which renders it susceptible to nuclease degradation. [0040] In a particularly preferred embodiment, the oligonucleotide scaffold has from 5 to 100 nucleic acid bases, more preferably from 10 to 40 nucleic acid bases. Similarly, the oligonucleotide factor is one which is substantially complimentary to the oligonucleotide scaffold and preferably has from 5 to 100 nucleic acid bases, more preferably from 10 to 40 nucleic acid bases. [0041] The skilled artisan will appreciate that the oligonucleotide constructs of the present invention may appear in a wide variety of formats, based on different combinations of bases and linkages.

Hybridization [0042] In a preferred embodiment, the oligonucleotide scaffold and the oligonucleotide factor have substantial, if not total, complementarity to each other over a significant portion of their respective sequences. This "significant portion" of the oligonucleotide sequences will typically exclude those parts of the sequences that are specifically designed not to be complementary, such as overhangs on either end of either oligonucleotide. These portions are generally included for functions other than oligonucleotide hybridization and release, such as attachment of diagnostic and therapeutic moieties, as will be described below. [0043] In the present invention, the two oligonucleotide sequences are substantially complementary if the two sequences hybridize to each other under stringent conditions. The phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which an oligonucleotide will hybridize to its target subsequence or a second oligonucleotide, typically in a complex mixture of nucleic acids, but not to other sequences lacking sufficient complementarity, or similarity to the target sequences. [0044] One of skill in the art will recognize that stringent conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The T_m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the oligonucleotides complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the oligonucleotides are occupied at equilibrium). [0045] The artisan will appreciate that stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C for short oligonucleotides (e.g., 10 to 50 nucleotides) and at least about 60° C for long oligonucleotides (e.g., greater than 50 nucleotides). [0046] It is known in the art that stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes.

[0047] Preferably, the oligonucleotide scaffold and the oligonucleotide factor are at least 50% complementary, more preferably at least 70% complementary, and most preferably at least 90% complementary along their target sequences. Again, this usually will exclude those portions of either oligonucleotide that are deliberately designed to not hybridize, such as overhands at the ends of either oligonucleotide.

Degradability [0048] In a preferred embodiment, the oligonucleotide scaffold can be essentially any oligonucleotide that degrades at a faster rate than does the oligonucleotide factor, under the same or similar conditions. In other words, the oligonucleotides are differentially degradable. [0049] In another preferred embodiment, the oligonucleotide scaffold is degraded by either an endonuclease or an exonuclease. Evaluation of nuclease degradation can be carried out according to established methods (see, for example, Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED., Cold Spring Harbor, NY, 1989), or by the an assay of the present invention (see Assay section).

[0050] In a preferred embodiment, the scaffold-factor complex of the present invention will exhibit an optimal combination of differentially degradable oligonucleotides. When evaluated in an assay for general nuclease activity, the degradation rate of the scaffold will preferably be 1.5 times the degradation rate of the factor. In a preferred embodiment, the degradation rate of the scaffold will be 2 times the degradation rate of the factor. In another preferred embodiment, the degradation rate of the scaffold will be 10 times the degradation rate of the factor. [0051] The artisan will recognize that it may be desirable to select oligonucleotide sequences that degrade at a faster rate under certain conditions, or alternatively degrade at a slower rate under certain conditions. Detection and evaluation of oligonucleotide degradability can be carried out according to established methods, or by an assay of the present invention (see Assay section). [0052] The artisan will appreciate that an oligonucleotide may be designed to exhibit a broad range of degradability characteristics. In a preferred embodiment, the oligonucleotide may include a modified base at one end, such as 3 '-3' thymidine, which is known to confer exonuclease resistance. [0053] In a preferred embodiment, the oligonucleotide includes phosphate linkages, which confer a certain degree of degradability to the oligonucleotide. In a related embodiment, the oligonucleotide includes phosphorothiolate linkages, which confer another degree of degradability. In another embodiment, the oligonucleotide includes methylphosphonate linkages, which confer yet another degree of degradability to the oligonucleotide. In still another embodiment, the oligonucleotide includes a phosphodiester linkage. In another embodiment, the oligonucleotide includes a peptide linkage.

[0054] Certain types of linkages are known to render an oligonucleotide either more, or less, susceptible to degradation. Some types of linkages are known to confer endonuclease susceptability and some types of linkages are known to confer exonuclease susceptability. One of skill in the art will appreciate that by combining various types of linkages, with various types of bases, it is possible to obtain oligonucleotides displaying a myriad of degradability profiles. [0055] The artisan will recognize that different degradability profiles will confer certain release mechanism kinetics, and these parameters may be optimized depending on the requirements of the therapy or assay.

[0056] In yet another embodiment, oligonucleotides may be assembled by "stacking" strands that hybridize via strand overhangs, so that three or four or more separate strands can all assemble together until release is initiated. [0057] In another preferred embodiment, the oligonucleotide scaffold is degraded by electromagnetic radiation, such as ultraviolet light, for example.

Therapeutic Active Agents [0058] In one group of embodiments, the oligonucleotide factor has an attached therapeutic agent. In this aspect of the invention, the therapeutic agent can be essentially any therapeutic agent that is capable of forming a covalent or non-covalent attachment to an oligonucleotide or to a linking group which is attached to an oligonucleotide. In some embodiments, the therapeutic agent will be modified to provide a non-interfering functional group which can serve as a point of attachment to the linking group or, more directly, to the oligonucleotide factor.

[0059] In a preferred embodiment, the therapeutic agent may be chosen from a group including small organic molecules, metals (often present as complexes of their ionic forms), peptides, nucleic acids, and in particular, known therapeutic agents (see, e.g., GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ninth Ed. Hardman, et al., eds. McGraw-Hill, (1996)).

[0060] In a related embodiment, the therapeutic agent is a metal present as a complex in its ionic form, and aids in the transport of DNA into cells. [0061] Preferred therapeutic agents for use in the present invention are selected from antibacterial agents, antiviral agents, antiproliferative agents, antifungal agents, immunosuppressive agents, analgesics and the like.

[0062] Suitable antibacterial agents include, but are not limited to, cefotaxime, ceftriaxone, rifampin, minocycline, ciprofloxacin, norfloxacin, erythromycin, vancomycin, amoxacillin, nafcillin, oxacillin, penicillin, ampicillin, and their related derivatives.

[0063] Antiviral agents useful in the present invention can be selected from idoxuridine, sorivudine, trifluridine, valacyclovir, cidofovir, acyclovir, famciclovir, ganciclovir, foscarnet, didanosine, stavudine, zalcitabine, zidovudine, ribavirin and rimantatine. [0064] Similarly, suitable antimicrobial agents (some of which are also known as antibacterial agents) can be selected from, for example, sulfanilamide, sulfamethoxazole, sulfacetamide, sulfisoxazole, sulfadiazine, penicillins (e.g., penicillins G and V, methicillin, oxacillin, naficillin, ampicillin amoxacillin, carbenicillin, ticarcillin, mezlocillin and piperacillin), cephalosporins (e.g., cephalothin, cefaxolin, cephalexin, cefadroxil, cefamandole, cefoxitin, cefaclor, cefuroxine, loracarbef, cefonicid, cefotetan, ceforanide, cefotaxime, cefpodoxime proxetil, ceftizoxime, cefoperazone, ceftazidime and cefepime), aminoglycosides (e.g., gentamycin, tobramycin, amikacin, netilmicin, neomycin, kanamycin, streptomycin, and the like), tetracyclines (e.g., chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline and minocycline), and macrolides (e.g., erythromycin, clarithromycin, azithromycin). [0065] In a particularly preferred embodiment, the therapeutic agent is an anticancer agent. Suitable anticancer agents of the present invention include cytotoxic antitumor antibiotics such as calicheamicin. In another embodiment, the therapeutic agent is paclitaxel (Taxol). The artisan will appreciate that the compositions and methods of the present invention are well suited to incorporate a wide variety of chemotherapeutic and cytotoxic agents. [0066] In another preferred embodiment, the therapeutic agent of the present invention is an immunoactive agent. A variety of immunoactive agents can be used in the invention including, for example, the interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,

IL-9, IL-10, IL-11, IL-12, IL-13, and IL-14), TNFα and γ-interferon. [0067] In another preferred embodiment, the therapeutic agent of the present invention is a peptide or protein. In a preferred embodiment, the peptides of the present invention is a zinc finger protein. In another embodiment, the protein of the present invention is vascular endothelial growth factor. [0068] In another preferred embodiment, the therapeutic agent of the present invention is a nucleic acid. In a particularly preferred embodiment, the present invention provides for the controlled release of a nucleic acid, with the purpose of replacing or augmenting the deficient genetic material of a diseased individual. For example, the methods and compositions of the present invention may be used to provide a novel vehicle for delivering correct copies of the nucleic acid that is deficient in cystic fibrosis patients.

[0069] In related embodiments, the therapeutic agent is a cosmoceutical, such as glycolic acid.

[0070] In related embodiments, the therapeutic agent is a nutraceutical, such as glucosamine or chondroitin sulfate. [0071] In a preferred embodiment, the therapeutic agent is an antibody, or a fragment thereof. In a particularly preferred embodiment, the antibody is directed toward beta amyloid peptide, which has been implicated in Alzheimer's disease. In another embodiment, the antibody is directed to cell surface receptors known as alpha-4-beta (NLA-4) or alpha-4-beta-7, which have been implicated in a range of inflammatory and non-inflammatory diseases, including Crohn's Disease and Multiple Sclerosis.

[0072] In a preferred embodiment, the therapeutic agent is a vaccine. In a related embodiment, the therapeutic agent is a vaccine for measles, mumps, polio, varicella, diptheria, hepatitis A, pertussis, tetanus, or haemophilus influenzae b. [0073] In another group of embodiments, the oligonucleotide factor itself is the therapeutic agent. In a particularly preferred embodiment, the oligonucleotide factor is an antisense oligonucleotide. It is known in the art that antisense molecules have demonstrated clinical efficacy for inhibiting the expression of certain viral proteins necessary for the viral replication. For example, it has been demonstrated that antisense oligonucleotides can inhibit the expression of hepatitis C virus proteins required for replication of the virus. [0074] In a preferred embodiment, the oligonucleotide factor is a gene therapy agent. The first disease approved for treatment with gene therapy was adenosine deaminase (ADA) deficiency, a rare genetic disease. Patients with this condition do not have normal ADA genes, and their defective genes do not produce the functional ADA enzyme. The result is severe immunodeficiency. Utilizing the compositions and methods of the present invention, the oligonucleotide factor may, for example, include a normal copy of the ADA, and thereby be used to treat ADA patients, and other patients suffering from genetic diseases. [0075] In another preferred embodiment, the oligonucleotide factor is a ribozyme. In a particularly preferred embodiment, the oligonucleotide factor is a ribozyme that can recognize, bind, and digest a disease-causing mRNA sequence. In one embodiment, the ribozyme confers anti-tumor activity by inhibiting the formation of vascular endothelial growth factor. In another embodiment, the ribozyme confers antiviral activity by interfering with viral replication.

Attachment of Active Agent [0076] Methods for attaching active agents with oligonucleotides are well known in the art and depend on the nature of the functional group used in the active agent as a site of attachment, as well as the site of attachment on the oligonucleotide. The linking group is a moiety which covalently links the oligonucleotide portion of the conjugate to the therapeutic agent, preferably through a chain of no more than 30 atoms, more preferably about 15 atoms. Additionally, the site of attachment on the oligonucleotide will preferably be either the 3' or 5' end of the oligonucleotide. In other embodiments, however, attachment to the oligonucleotide can be to a nucleotide in an intermediate or interior position, and particularly to the heterocyclic base of the nucleotide in an intermediate position. [0077] In a presently preferred embodiment, the linking group is derived from a bifunctional molecule so that one functionality such as an amine functionality is attached, for example, to the phosphate on the 5' end of the oligonucleotide, and the other functionality such as a carbonyl group (CO) is attached to an amino group or hydroxy group of the therapeutic agent. Heterobifunctional linking groups are available from a number of commercial sources including, for example, Pierce Chemical Co. (see 1999/2000 Catalog pages 173-209 directed to cross-linking agents) and Biosearch Technologies.

[0078] In a related embodiment, the linking group may be derived from an amino alcohol so that the alcohol function is linked, for example, to the 3'-phosphate end of the oligonucleotide and the amino function is linked to a carbonyl group of the therapeutic agent. Yet another linking group includes an aminoalcohol (attached to the 3'-phosphate with an ester linkage) linked to an aminocarboxylic acid which in turn is linked to a therapeutic agent. In preferred embodiments, the linking groups can be represented by the formulae: -NH(CH₂)_mCO-; -O(CH₂)_mCO-; -O(CH₂)_mNH-; and -O(CH₂)_mCH(OH)(CH₂)_nNHCO(CH₂)_pNH-; wherein each of the subscripts m, n and p is independently an integer of from 1 to about 20 with the proviso that the overall length of the linker in no more than 30 atoms, and preferably no more than about 15 atoms. Preferred embodiments of these linking groups are: - NH(CH₂)₅CO-; -O(CH₂)_mCO-; -O(CH₂)₆NH-; and -OCH₂CH(OH)CH₂NHCO(CH₂)₂NH-

[0079] In some embodiments, the linking group will be one that is cleaved in vivo to release the therapeutic agent. Accordingly, the linking group can have internal functional groups (e.g., -SS-, -COO-, or -CONH-) that can be cleaved, for example, by glutathione, esterases or proteases, to release the therapeutic agent from the oligonucleotide. Related technology is described in, for example, PCT application US98/10571 (WO 98/52614). [0080] In one particularly preferred embodiment of the present invention, the therapeutic agent is covalently attached to the factor oligonucleotide, and the scaffold and factor oligonucleotides each comprise from 5 to 50 bases and are at least 90% complementary, the scaffold oligonucleotide having a faster degradation rate that the degradation rate of the factor oligonucleotide, when contacted with an endonuclease under the same or similar conditions.

[0081] In one particularly preferred embodiment of the present invention, the therapeutic agent is covalently attached to the factor oligonucleotide, and the scaffold and factor oligonucleotides each comprise from 5 to 50 bases and are at least 90% complementary, the scaffold oligonucleotide having a faster degradation rate that the degradation rate of the factor oligonucleotide, when contacted with an exonuclease under the same or similar conditions.

Alternative Oligonucleotide Constructs [0082] In one group of embodiments, the scaffold-factor complex is in the form of a DNA gel. The use of longer DNA as a viscosity agent to form a suitable gel has been described in PCT publication WO 01/24775. Accordingly, in this group of embodiments, the gel-forming DNA can also serve as the scaffold oligonucleotide which, upon nuclease degradation, releases oligonucleotide factors that can have attached therapeutic agents. Alternatively, degradation of the DNA gel can release an oligonucleotide factor that is itself, a therapeutic agent. In still other embodiments, the DNA gel (scaffold) contains additional therapeutic agents that are not attached to an oligonucleotide factor, yet can provide therapeutic benefit when released at a desired site with an oligonucleotide factor having attached therapeutic agents. [0083] In another preferred embodiment, the oligonucleotide factor includes a string of oligonucleotide subsegments, either covalently joined in a linear fashion, or as discrete oligonucleotide factors. In u s embodiment, each of the oligonucleotide subsegments can be linked with an active agent. This approach provides another mechanism by which the amount of active agent released by the composition may be modulated or otherwise controlled. For example, by increasing the number of active agents on an oligonucleotide factor, or by providing multiple oligonucleotide factors along a single oligonucleotide scaffold, it is possible to release etiher higher concentration of active agent, or a "timed- release" of active agent to a desired location. In the case of the "timed-release" approach, the oligonucleotide scaffold will, in preferred embodiments, be selected to be exonuclease sensitive. That is, the oligonucleotide scaffold will be designed to be degraded from its terminus, thereby releasing oligonucleotide factor (with or without an attached therapeutic agent) at a determinable rate. [0084] In related embodiments, the present invention provides methods for the controlled release of a therapeutic agent from a scaffold- factor complex, wherein the scaffold-factor complex is administered to a subject in combination with a suitable exonuclease. In one group of particularly preferred embodiments, the oligonucleotide scaffold portion of the scaffold-factor complex is attached at one end to a solid support and is designed to be exonuclease susceptible. Preferably, the complex comprises a plurality of oligonucleotide factor/therapeutic agent moieties. More preferably there are from 2 to 100 oligonucleotide factor/therapeutic agent moieties associated with each oligonucleotide scaffold.

Targeting Moieties [0085] To facilitate the targeted delivery of the present compositions, the composition can also have a covalently or non-covalently attached targeting moiety. A variety of targeting moieties are useful in the compositions described herein and can be any group that makes it possible to direct the transfer of the scaffold-factor complex a particular site. The targeting moiety can be an extracellular targeting agent, which allows, for example, the complex to be directed towards certain desired tissues or certain types of cells, such as tumor cells, liver cells, hematopoietic cells, and the like. Such a targeting moiety can also be an intracellular targeting agent, allowing the complex to be directed towards particular cell compartments or components, such as mitochondria, nucleus, and the like.

[0086] In a preferred embodiments, the targeting moiety can be attached to either the oligonucleotide scaffold, or to the oligonucleotide factor. [0087] The targeting moiety of the present invention may be chosen to direct the scaffold- factor complex to a location where the factor may be released upon nuclease triggered degradation. In another embodiment, the targeting moiety is attached with the oligonucleotide factor itself, to allow for more specific uptake of the oligonucleotide after release.

[0088] In a presently preferred embodiment, the targeting moiety or moieties are linked covalently to the scaffold-factor complex, optionally via a linking group. Methods of attaching targeting moieties, as well as other biological agents, to nucleic acids are well known to those of skill in the art using, for example, heterobifunctional linking groups. In one group of embodiments, the targeting moiety is a fusogenic peptide for promoting cellular transfection, that is to say for favoring the passage of the scaffold- factor complex across membranes, or for helping in the egress from endosomes or for crossing the nuclear membrane. In a related embodiment, the targeting moiety is a cationic peptide. [0089] In another embodiment, the targeting moiety can also be a cell receptor ligand for a receptor that is present at the surface of the cell type, such as, for example, a sugar, transferrin, insulin or asialo-orosomucoid protein. Such a ligand may also be one of intracellular type, such as a nuclear location signal (nls) sequence which promotes the accumulation of transfected DNA within the nucleus. [0090] Other targeting moieties useful in the context of the invention, include sugars, peptides, hormones, vitamins, cytokines, oligonucleotides, lipids or sequences or fractions derived from these elements and which allow specific binding with their corresponding receptors. Preferably, the targeting moieties are sugars and or peptides such as antibodies or antibody fragments, cell receptor ligands or fragments thereof, receptors or receptor fragments, and the like. More preferably, the targeting moieties are ligands of growth factor receptors, of cytokine receptors, or of cell lectin receptors or of adhesion protein receptors. [0091] In yet another embodiment, the targeting moiety can be a sugar, which makes it possible to target lectins such as the asialoglycoprotein receptors, or alternatively an antibody Fab fragment which makes it possible to target the Fc fragment receptor of immunoglobulins. The term "antibody", as used herein, means an immunoglobulin, whether natural or wholly or partially synthetically produced. All derivatives thereof which maintain specific binding ability are also included in the term. The term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgG class, however, are preferred in the present invention. Additionally, the term "antibody fragment" refers to any derivative of an antibody which is less than full-length. Preferably, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')₂, scFv, Fv, dsFv diabody, and Fd fragments. The antibody fragment may be produced.by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively, the antibody fragment may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfϊde linkages. The fragment may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.

[0092] In one group of particularly preferred embodiments, the targeting moiety is an antibody or antibody fragment having specificity for a cell surface receptor. Like the therapeutic agents, the antibody or antibody fragment can be attached by means of a linking group to either terminous of either oligonucleotide in the composition. Preferably, the targeting agent is attached to the oligonucleotide scaffold, and is released from the composition as the oligonucleotide scaffold is degraded. More preferably, the targeting moiety is attached to a site in the oligonucleotide scaffold which is spatially removed from the attached therapeutic agent so that the targeting moiety is free to interact with its binding partner. In a related embodiment, the binding partner is a cell surface antigen. [0093] For example, in those embodiments in which the therapeutic agent is attached to the 5 '-end of the oligonucleotide factor, the targeting moiety will preferably be attached to the 5 '-end of the oligonucleotide scaffold. Alternatively, for those embodiments in which the therapeutic agent is attached to the 3 '-end of the oligonucleotide factor, the targeting moiety will preferably be attached to the 3 '-end of the oligonucleotide scaffold. [0094] In a particular example, the targeting moiety is directed to a cell surface receptor such as the CD33 antigen, a sialic acid-dependent adhesion protein found on the surface of leukemic blasts and immature normal cells of myelomonocytic lineage, but not on normal hematopoietic stem cells.

Diagnostic Active Agents [0095] In a preferred embodiment, the methods and compositions of the present invention may further include a "label" or a "detectable moiety." These groups include a component that is detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, for example, by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide. [0096] A "labeled" oligonucleotide is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the oligonucleotide may be detected by detecting the presence of the label bound to the oligonucleotide. [0097] The attachment may be covalent or non-covalent binding, but the method of attachment is not critical to the present invention. The label allows the labeled oligonucleotide to produce a detectable signal that is related to the presence of the labeled oligonucleotide. The label is selected to directly bind to the oligonucleotide or to indirectly bind the oligonucleotide by means of an ancillary binding member. The label can be incorporated into either the oligonucleotide scaffold or the oligonucleotide factor, or both, depending on the format of the invention. [0098] In a preferred embodiment, a diagnostic agent may be attached to the oligonucleotide scaffold, the oligonucleotide factor, or both.

[0099] In a presently preferred embodiment, a label refers to any substance which is capable of producing a signal that is detectable by visual or instrumental means. Various labels suitable for use in the present invention include labels which produce signals through either chemical or physical means. Such labels can include enzymes and substrates, chromogens, catalysts, nanoparticles, nanocrystals, quantum dots, fluorescent compounds and proteins, chemiluminescent compounds, and radioactive labels. Other suitable labels include colloidal metallic particles such as gold, colloidal non-metallic particles such as selenium or tellurium, dyed or colored particles such as a dyed plastic or a stained microorganism, organic polymer latex particles and liposomes, colored beads, polymer microcapsules, sacs, erythrocytes, erythrocyte ghosts, or other vesicles containing directly visible substances, and the like.

[0100] In a preferred embodiment, a visually detectable label is used, thereby providing for the direct visual or instrumental readout of the presence or amount of the oligonucleotide in the test sample without the need for additional signal producing components. [0101] The selection of a particular label is not critical to the present invention, but the label will be capable of generating a detectable signal either by itself, or be instrumentally detectable, or be detectable in conjunction with one or more additional signal producing components, such as an enzyme/substrate signal producing system. It will be appreciated by one skilled in the art that the choice involves consideration of the oligonucleotide to be detected and the desired means of detection. A label may also be incorporated in a control system for the assay.

[0102] In a preferred embodiment, one or more signal producing components can be reacted with the label to generate a detectable signal. If the label is an enzyme, then amplification of the detectable signal is obtained by reacting the enzyme with one or more substrates or additional enzymes and substrates to produce a detectable reaction product. [0103] In a related embodiment, the label can be a fluorescent compound where no enzymatic manipulation of the label is required to produce the detectable signal. Fluorescent molecules such as fluorescein, phycobiliprotein, rhodamine and their derivatives and analogs are suitable for use as labels in such an embodiment.

[0104] The use of dyes for staining biological materials, such as proteins, carbohydrates, nucleic acids, and whole organisms is documented in the literature, and are contemplated by the compositions and methods of the present invention. It is known that certain dyes stain particular materials preferentially based on compatible chemistries of dye and ligand. For example, Coomassie Blue and Methylene Blue for proteins, periodic acid-Schiff s reagent for carbohydrates, Crystal Violet, Safranin O, and Trypan Blue for whole cell stains, ethidium bromide and Acridine Orange for nucleic acid staining, and fluorescent stains such as rhodamine and Calcofluor White for detection by fluorescent microscopy. Further examples of labels can be found in, at least, U.S. Patent Nos. 4,695,554; 4,863,875; 4,373,932; and 4,366,241, all incorporated herein by reference.

[0105] In still another embodiment, the released oligonucleotide factor itself can code for a fluorescent or otherwise detectable protein (e.g, green fluorescent protein). [0106] One of skill in the art will appreciate that a wide variety of diagnostic imaging and contrast agents maybe used as the active agent of the present invention. Further, it will be recognized that the diagnostic agent can be detected in vivo by invasive or non-invasive means. In a preferred embodiment, the diagnostic agent is a fluorescent or radioactive molecule, or a gadolinium contrast agent that can be images or visualized intraoperatively. Pharmaceutical Formulations [0107] In another aspect, the present invention provides a composition and method for the controlled release of an active agent in a subject, wherein a composition is administered to a subject or patient. The composition typically comprises a pharmaceutically acceptable carrier and a scaffold-factor complex as described in detail above. Briefly, this scaffold-factor complex includes an oligonucleotide scaffold and a substantially complementary oligonucleotide factor. The oligonucleotide factor has an attached active agent, or is an active agent itself. Alternatively, the scaffold can have attached active agents as well. The two oligonucleotides are differentially degradable, so that degradation of the scaffold occurs a faster rate than the degradation of the factor. As the scaffold degrades, the factor is released.

[0108] As noted above, the composition further includes a pharmaceutically acceptable carrier or excipient. The nature of the excipient will depend largely on the method of administration.

[0109] The compositions of the present invention can be prepared and administered in a wide variety of oral, topical and parenteral dosage forms. Thus, the compositions of the present invention can be administered by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally. Also, the compositions described herein can be administered by inhalation, for example, intranasally. Additionally, the compositions of the present invention can be administered transdermally. [0110] For preparing pharmaceutical compositions from the scaffold-factor complexes described above, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

[0111] In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. [0112] In a group of embodiments, the powders and tablets preferably contain from 5% or 10% to 70% of the active complex. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active complex with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration. [0113] For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify. [0114] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution. [0115] Aqueous solutions suitable for oral use can be prepared by dissolving the active complex in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active complex in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

[0116] Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active complex, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

[0117] The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active complex. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. [0118] The quantity of active complex in a unit dose preparation may be varied or adjusted from 0.1 mg to 1000 mg, preferably 1.0 mg to 100 mg for traditional pharmaceutical agents, according to the particular application and the potency of the active component. The composition can, if desired, also contain other compatible active agents. In some embodiments, the amount of active complex in a unit dose preparation can be as little as 0.1 picograms up to about 100 nanograms, depending on the nature of the therapeutic agent and its desired effect.

[0119] In therapeutic use for the treatment of bacterial infections, the compounds utilized in the pharmaceutical method of the invention are administered at the initial dosage of about 0.001 mg/kg to about 100 mg/kg daily. A daily dose range of about 0.1 mg/kg to about 10 mg/kg is preferred. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.

Release Of Active Agent From Solid Support [0120] In a preferred embodiment, the present invention provides a method and composition for the controlled release of an active agent from a solid or semisolid support. The composition includes an oligonucleotide scaffold and a substantially complementary oligonucleotide factor. The scaffold is attached with a solid support. An active agent is attached to the oligonucleotide factor. The two oligonucleotides are differentially degradable, so that degradation of the scaffold occurs a faster rate than the degradation of the factor. As the scaffold degrades, the factor is released from the support.

[0121] One of skill in the art will appreciate that the scaffold may be covalently or noncovalently attached with the support by a wide spectrum of mechanisms. There are many therapeutic and diagnostic applications for the support-based release of an oligonucleotide factor. [0122] A variety of solid or semisolid supports are useful in the present invention. In one group of embodiments, the support is a semisolid support such as a gel or hydrogel composition. See, for example, co-owned and co-pending application Ser. No. 09/675,566 entitled "Gel-Forming Compositions" filed September 29, 2000, which describes one group of semisolid supports useful for the controlled release of the therapeutic agent or oligonucleotide composition. Still other supports are described in U.S. Patent Nos. 5,879,713; 5,858,746; 5,919,484; and 5,514,379.

[0123] In another group of embodiments, the support is a solid support, such as an implantable biosensor, a stent, or a diagnostic assay device. Suitable supports are described in, for example, Von Recum's Handbook of Biomaterials Evaluation : Scientific, Technical, and Clinical Testing of Implant Materials, Hemisphere Publishing, 2^nd Ed. Nov. 1998. In a related embodiment, the support is a stent, and the oligonucleotide factor is an antisense molecule configured to inhibit smooth muscle cell proliferation.

[0124] In other embodiments, the support is a medical device such as an embolization coil, an orthopedic prosthetic, or an implantable device for drug delivery. In a particularly preferred embodiment, the scaffold is attached to the polymeric backbone of a biodegradable gel used to deliver an active agent. Optionally, the support may be a synthetic or natural tissue graft which is pre-treated by the composition or method of the present invention, thereby reducing or preventing infection or disease, or modulating a graft-host immune response. In a related embodiment, the support is a three dimensional scaffold for tissue engineering.

[0125] The artisan will appreciate that many materials are suitable for attachment with the scaffold. Among these are glass, gold, device grade polymers such as PTFE, stainless steel, and the like. Kits for Controlled Release Compositions

[0126] In a further preferred embodiment, the present invention provides a kit for the preparation of a controlled release composition. The kit includes a first container holding an oligonucleotide scaffold, and a second container holding a substantially complementary oligonucleotide factor. An active agent is attached to the oligonucleotide factor. The two oligonucleotides are differentially degradable, so that degradation of the scaffold occurs a faster rate than the degradation of the factor. In use, as the scaffold degrades, the factor is released.

Assays functional genomics [0127] In a preferred embodiment, the compositions and methods of the present invention are configured for functional genomic assays. In a particularly preferred embodiment, the oligonucleotide factor is an antisense molecule. In another particularly preferred embodiment, the oligonucleotide factor is a ribozyme molecule. Because these oligonucleotide-based molecules are able to specifically inhibit the expression of one gene at a time, without knowing anything more than the DNA sequence of the gene, they are very useful in helping to determine the function of genes in different cell types. It is this translation of genomic information into functional information that is the hallmark of functional genomics. The functional genomics approach is well adapted for identifying specific gene candidates for drug targets, among the tens of thousands of genes present in the human genome.

[0128] In a presently preferred embodiment, a ribozymal oligonucleotide factor is released from a cell culture plate, wherein the factor is configured to inhibit gene expression in cells grown on the plate.

[0129] In still another embodiment, an array of tethered scaffold- factor complexes are provided on a support. Each complex is assigned to a known location, and each complex includes an oligonucleotide factor having a known sequence. A myriad of distinct sequences can be represented by the oligonucleotide factors. A culture of cells is then deposited or grown on the support. As nuclease is added to the culture, the scaffold strands are degraded, and the factor strands are released. The released antisense or ribozyme in each location will then specifically inhibit its particular gene only in the overlying cells growing in that vicinity. Because many different oligonucleotide factors are being released from many different locations on the on the support, it is possible to study the function of many genes on the cells at once. The properties of the antisense-treated cells can then be analyzed in detail, such as immunostaining for protein expression, and in parallel with their neighbors, with known locations on the support corresponding to the gene being studied.

[0130] The artisan will appreciate that this approach to functional genomics is well adapted for high throughput assays, as the locations can be made very small, and therefore fewer cells are required to maximize the number of genes studied at once.

[0131] In another preferred embodiment, a ribozymal or antisense oligonucleotide factor is introduced in a mammalian model, such as a mouse or a rat, for in vivo assays. In this embodiment, the factor is configured to inhibit gene expression in certain cells of the model. The artisan will appreciate that the scaffold-factor complex may be introduced directly into the model, or the complex may be introduced while attached to a solid or semisolid support. [0132] In yet another preferred embodiment, the methods and compositions of the present invention are adapated for use in a cell-selectivity assay. In this embodiment, the scaffold strand includes a cell-type specific antibody targeting moiety, and the factor strand is an antisense oligonucleotide that includes a fluorescent marker. This scaffold- factor complex is then contacted with a mixed cell culture containing a first cell type having antibody specificity, and a second cell type lacking the antibody specificity. Once the scaffold-factor complexes are allowed to bind to the appropriate cells, a nuclease such as exoIII is added to initiate degradation of the scaffold strand. Subsequently, the labeled antisense factor releases from the scaffold, is taken up into the cells, and exerts its antisense effect. All cells are then stained to determine whether the cells coated with the antibody, the cells exhibiting fluorescence, and the cells with modulated mRNA or protein expression of the antisense-affected gene are all of the same cell type. degradation assays [0133] In a preferred embodiment, the present compositions and method may be used in an assay to detect the presence of a degradative agent, or to measure the activity of a degradative agent. As the oligonucleotide factor is release from the scaffold in the presence of a degradative agent, the detection or measurement of the released oligonucleotide factor can serve as an assay for the degradative agent. In a particular embodiment, the degradative agent is a nuclease. In a related embodiment, the degradative agent is electromagnetic radiation.

[0134] In a particularly preferred embodiment, the present compositions and methods may be used in an assay to detect the presence of a nuclease. Since the oligonucleotide factor is released from the scaffold in the presence of an appropriate nuclease, the detection of the released oligonucleotide factor can itself serve as a detection mechanism for the presence of that nuclease. In a related embodiment, this assay is used to confirm that a sample is substantially free of nuclease, or that a sample affirmatively contains a nuclease. [0135] In a related embodiment, an assay is provided for measuring the nuclease activity in a test sample. In a related embodiment, the test sample may include a compound with known or suspected nuclease activity.

[0136] The artisan will recognize that many different types of body fluids may contain varying levels of nuclease activity, and it is of interest to detect or quantify these nuclease levels. In a particularly preferred embodiment, the assay of the present invention is used to determine the nuclease activity of a body fluid. In related embodiments, the body fluid can be serum, joint fluid, cerebrospinal fluid, sweat, saliva, pleural effusion, ascites, extracellular fluid (ECF) or various tumors, ECF of ischemic and necrotic tissues, or ECF in acutely and chronically inflamed tissues. Similarly, the fluid may be intracellular fluid of any cell type under normal, abmormal, necrotic, or apoptotic conditions, including cytoplasmic or lysozomal fluid. [0137] Those of skill in the art will recognize that the assays of the present invention are well suited for characterizing either exonuclease activity or endonuclease activity. oligonucleotide assays [0138] In a preferred embodiment, the compositions and methods of the present invention are configured to assay the degradability of certain scaffold sequences. The artisan will appreciate that in many cases, it is desirable to detect or measure the degradability of a test oligonucleotide sequence. Test sequences may be used as oligonucleotide scaffolds, and treated with various degradation protocols. Detectable oligonucleotide factors can be used to aid in the determination of whether the scaffold is degradable, or to what degree the scaffold is degradable. This assay approach can be used to characterize the degradability profiles of certain oligonucleotide sequences and constructs. In preferred embodiments, these assays are used to determine the effect that certain linkages, bases, capping or blocking groups, and other attached active agents have on the degradability of an oligonucleotide. other assays [0139] In yet another embodiment, the present invention provides a in vitro diagnostic assay, where the released oligonucleotide factor can be used to detect or remove a specific oligonucleotide sequence in an unknown sample containing multiple oligonucleotide strands.

[0140] The present invention is particularly suitable for a test device as shown in the accompanying drawings and examples. It is understood that the drawings and examples are provided for purposes of illustration and not meant limit the scope of the present invention.

EXAMPLES

[0141] The invention can be better understood by way of the following examples which are representative of certain preferred embodiments, but are not to be construed as limiting the scope of the invention.

EXAMPLE 1

[0142] This example illustrates the DNA-mediated controlled release of DNA oligonucleotides.

[0143] Two DNA oligonucleotides each labeled with fluorescein (a fluorescent marker) at the 5' end were obtained from Sigma Genosys. The scaffold strand A was 27bp long and unmodified at the 3¹ end, and hence susceptible to exonuclease at this end. The factor strand B was 18bp long and capped at the 3' end with a 3'-3' thymidine base to confer exonuclease resistance at this end. Strand B was complementary to bases 10 through 27 on strand A, and hence hybridized perfectly to strand A when both were put in solution under annealing conditions. The A:B duplex was examined in the presence and the absence of exonuclease. [0144] FIG. 5 is a polyacrylamide gel showing that when nuclease was absent, the A:B complex remained stable in size and quantity over time (lanes 1-3). In addition to the band corresponding to the heavier scaffold-factor complex, a second band is visible, corresponding to an excess of unbound scaffold strand A.

[0145] In the presence of exonuclease, the A:B complex became smaller and initially more variable in size, signifying the gradual degradation of scaffold strand A in the complex. The degradation of the excess scaffold strand A is also clearly observable. Over time, increasing amounts of an 18bp band, corresponding to strand B by itself, appeared in the samples, signifying released oligonucleotide(lanes 4-9). Eventually, only strand B of the complex remained, signifying completion of release (lane 10). Lanes 11 and 12 are control samples of strand B, and strand A, respectively. This result indicates that nuclease activity can trigger gradual release of a nuclease-resistant oligonucleotide from a hybridizing nuclease-susceptible scaffold DNA strand in a controlled release fashion.

EXAMPLE 2 [0146] This example illustrates the controlled release of an active agent from a solid support.

Materials

[0147] An unmodified 27-mer oligonucleotide sequence, 5'-AAAAAAAAATTTGAGGCACGCCTGATC-3' (0.1 μg/μL) was obtained from Sigma Genosys (Woodlands, Texas, USA). An 18-mer oligonucleotide 5'-GATCAGGCGTGCCTCAAA-3' (0.1 μg/μL) was also obtained from Sigma Genosys with a terminal block (as described in Example 1) to provide exonuclease resistance and with FITC as a fluorescent label for visualization attached to the 5'-end. Exonuclease III (200 units/μL) and Exonuclease III 10X Reaction Buffer (E577A) were obtained from Promega (Madison, Wisconsin, USA). Fetal Bovine Serum was obtained from Gibco BRL (Gaithersburg, Maryland, USA). 99% Methylimidazole assay grade reagent was obtained from Research Chemicals Ltd (Heysham, GBR). EDC was obtained from Pierce (Rockford, Illinois, USA). Dnase Rnase free distilled water and TE (Tris EDTA) buffer pH 8.0 (sterile) were obtained from Gibco BRL.

Procedures

Derivatization of stainless steel surface.

[0148] Stainless steel serves as the material for a number of medical devices including vascular stents, which are in contact with blood (and thus serum). Since there is some interest in release of oligonucleotides from a stent, this model represents a medically relevant one for feasibility demonstration of the technologies described herein. With this context in mind, sheets of 316 stainless steel (McMaster-Carr Supply company, Atlanta, GA) were treated with allylamine in a plasma chamber to introduce reactive amine groups, as depicted in FIG. 6. To 1 cm pieces of allylamine-stainless steel surfaces, 980 μL of distilled water (rnase- and dnase-free), 10 μL of methylimidazole and 10 mg of EDC were added to activate the amines for reaction with the terminal phosphate of DNA as depicted in FIG. 6. The mixture was shaken vigorously for 10 seconds.

Preparation of27-mer oligo DNA for attachment [0149] 18 μL (0.18 μg) of 27-mer oligonucleotide DNA was added to a 1.5 mL microfuge tube and brought to 40 μL total volume with distilled rnase- and dnase- free water and mixed vigorously for about 15 seconds. The dna solution was then placed in a heating block and heated to 100°C for 5 min in order to separate the strands. The solution was then vortexed for about 10 seconds after the heating in order to create a uniform solution. Attachment of single stranded 27mer oligos to washers [0150] A total of 10 μL (0.45 μg) of the single stranded 27-mer oligonucleotides was added to each microfuge tube containing the activated stainless steel mixture from above. The microfuge tubes were then inverted twice and placed in an aluminum block and heated at 60°C overnight. The activated stainless steel pieces were then rinsed with TE buffer and stored in microfuge tubes containing TE buffer and refrigerated.

Annealing oflδmer oligos to 27mer oligos on activated stainless steel pieces [0151] The 27-mer oligonucleotide-treated activated stainless steel pieces in 1.0 mL TE were heated to 72°C (about 5°C above the melting temperature of the 18-mer oligos). Then, 4.4 microliters (0.44 micrograms) of 18mer oligos was then added to each microfuge containing the 27mer oligo activated stainless steel pieces. The mixture was vortexed for about 30 seconds and then heated at 72°C for 5 min in a heating block. Afterwards, the microfuge tubes were wrapped in foil to prevent quenching and placed in a 37°C water bath for approximately 30 min. The 18-mer oligos were complementary to the anchored 27-mer and would be expected to anneal under these conditions. Also, the 18-mer (unlike the 27- mer) was capped to provide exonuclease resistance. Finally, the 18-mer was linked to FITC as a fluorescent label which allowed visualization (through fluorescence or absorbance) and represented the possibility of anchoring an unrelated therapeutic to the DNA strand as well. Thus, this experiment demonstrated binding of DNA specifically for release either of a therapeutic DNA strand or a therapeutic agent bound to DNA. The tubes were stored at 4°C.

Analysis of DNA on activated stainless steel pieces [0152] The treated stainless steel pieces were analyzed using image analysis and FTIR of the surface. The treated stainless steel pieces were vortexed and each placed in a 50 mL centrifuge tube filled with phosphate buffer saline. The 50 mL tubes with stainless steel pieces were then vortexed for 30 seconds and taken out with forceps so as not to contaminate the surface. The stainless steel pieces were then blotted dry and placed under a UN lamp and an image of each side of the treated stainless steel pieces were taken. The images were then labeled "time 0" and the density of the fluorescence on the surface was analyzed using ΝIH image (version 1.62, ΝIH, Bethesda, MD) software with mean and standard error determined in Statview (Abacus Concepts, Berkeley, CA) and significance determined using one way AΝONA repeated measures with post-hoc testing using Scheffe F-test at 95%. Results are depicted in FIG. 7 (exonuclease III experiment) and FIG. 8 (serum experiment). [0153] Single pass attenuated total reflectance Fourier transform infrared spectroscopy (Thermo Nicolet, Madison, WI) was used to determine the quantity of DNA on the surface of the stainless steel pieces. The area under the curve from 1350-1700 was measured with mean and standard error determined as before and results summarized in FIG. 9 (exonuclease III experiment) and FIG. 10 (serum experiment).

Preparation of Exonuclease LLl and Fetal Bovine Serum [0154] General feasibility for release of capped 18mer oligo from the washer surface was analyzed through the use of exonuclease III. Because the 18mer oligo contained a 3' cap, only the 27mer oligo would be degraded. The degradation of the 27mer oligo would then enable a timed release of the 18mer oligo from the washer surface. A 1:50 dilution of exonuclease III solution was prepared in a 15 mL centrifuge tube by adding 100 μL of exonuclease III, 500 μL of exonuclease III buffer and brought to 5 mL with rnase- and dnase- free distilled water. Three microfuge tubes were accordingly labeled "60 min", "120 min", and "24 hr." 1 mL of the exonuclease III solution was then aliquotted to each of the labeled microfuge tubes and stored in the refrigerator overnight.

[0155] Biologically relevant release was evaluated by release of capped oligos in the presence of serum (which contains exonucleases) rather than high level exogenous exonuclease. For this experiment, a set of microfuge tubes was labeled "FBS-60min", "FBS- 120min" and "FBS-24hr." 1 mL of fetal bovine serum was aliquotted to each of the microfuge tubes. The microfuge tubes were then refrigerated overnight.

Release ofl8mer oligo from Washer Surface [0156] A treated washer with 18mer oligos annealed to 27mer oligos on the surface was added to each of the tubes labeled "60 min" with exonuclease III and "FBS-60min." The tubes were then wrapped in foil to prevent quenching of fluorescent label and placed in a 37°C water bath for 60 min. After 60 minutes, the washers were taken out the tubes and placed in 50 mL centrifuge tubes containing phosphate buffer saline. The microfuge tubes with the 60 min solutions were then wrapped in foil to prevent quenching of fluorescent labels and placed at 4°C for UV/Vis spectrophotometer analysis. The centrifuge tubes were then vortexed for 30 seconds. The washers were then blotted dry. Pictures of each side of the washer surface under a UV lamp were taken and analyzed for fluorescence density. FTIR analysis of the surface of the washers was also taken and the area under the curve from 1350 to 1700 wavelength was measured. Afterwards, the washer from the exonuclease III solution was placed into the microfuge labeled "120min" and placed in the 37°C water bath for one hour. The same was done for the washer from the FBS solution. After one hour, the washers were placed separately in a 50 mL centrifuge tube containing phosphate buffer saline and vortexed for 30 seconds. The washers were then analyzed the same way as the 60 min time point and the microfuge tubes labeled "120 min" were wrapped in foil and stored 4°C for UVVis analysis. The washers were then placed in the microfuge tubes labeled "24 hrs", the exonuclease III treated washer wentinto the tube containing exonuclease III and-the FBS- treated washer was placed into the microfuge containing FBS. The tubes were then placed into the 37°C water bath for 22 hours. The washers were then rinsed, and analyzed the same way as the 60 min and 120 min time points and the microfuge tubes labeled "24 hrs" were stored 4°C for UV-Vis analysis. The results from the experiment are summarized in FIG. 7 (exonuclease III via surface fluorescence), FIG. 8 (exonuclease III via FTIR), FIG. 9 (serum via surface fluorescence), and FIG. 10 (serum via FTIR). [0157] For FIG. 7, surface fluorescence at time zero was statistically significantly higher than at 60 minutes, 120 minutes and 24 hours of Exonuclease III exposure, and surface fluorescence 60 minutes was significant versus 120 minutes. However, there was no statistically significant difference between 120 minutes and 24 hours in surface fluorescence after treatment with Exonuclease III as described. Thus, exonuclease III can be used to give rapid specific release of the nondegradable strand from the derivatized surface. [0158] For FIG. 8, surface fluorescence at time zero was statistically significantly higher than at 60 minutes, 120 minutes and 24 hours of serum exposure, and surface fluorescence 60 minutes was significant versus 120 minutes and 24 hours. Additionally, there was statistical trending toward significance (defined as 0.08>P>0.05) between 120 minutes and 24 hours in surface fluorescence after treatment with serum as described. Thus, serum can be used to give timed specific release of the nondegradable strand from the derivatized surface. This example suggests that controlled nucleotide-based release of therapeutics from devices in contact with blood is immediately achievable.

[0159] For FIG. 9, surface FTIR absorbance at time zero was statistically significantly higher than at 60 minutes, 120 minutes and 24 hours of Exonuclease III exposure. However, there was no statistically significant difference from 60 minutes onward in surface fluorescence after treatment with Exonuclease III as described. Thus, exonuclease III can be used to give rapid specific release of the nondegradable strand from the derivatized surface. [0160] For FIG. 10, surface FTIR absorbance at time zero was statistically significantly higher than at 60 minutes, 120 minutes and 24 hours of serum exposure, and surface fluorescence 60 minutes was significant versus 120 minutes and 24 hours, and 120 minutes was significant versus 24 hours. Thus, serum can be used to give slower sustained specific release of the nondegradable strand from the derivatized surface.

[0161] Supernatants from the experiments above underwent scanning UN- Vis analysis in a Perkin-Elmer DU 640 to confirm presence of DΝA (OD 260) and label (OD495). IR was performed to confirm that DΝA was intact (i.e. nondegradable strand with label was released undegraded).

[0162] Overall, this experiment demonstrates that either in a controlled fashion (exogenous exonuclease) or in a biologically relevant fashion (serum), specific nucleotide release can be achieved with cleavage of complementary nucleotide segments.

[0163] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A composition for the controlled release of an active agent, said composition comprising: (a) an oligonucleotide scaffold; (b) an oligonucleotide factor, said scaffold and factor oligonucleotides being substantially complementary; and (c) an active agent attached to the oligonucleotide factor; wherein the rate at which the scaffold degrades is greater than the rate at which the factor degrades.

2. A composition in accordance with claim 1, wherein said scaffold and factor oligonucleotides each comprise from 5 to 100 nucleic acid bases.

3. A composition in accordance with claim 1, wherein said scaffold and factor oligonucleotides each comprise from 10 to 40 nucleic acid bases.

4. A composition in accordance with claim 1, wherein said active agent is covalently attached to said oligonucleotide factor.

5. A composition in accordance with claim 1, wherein the rate at which the scaffold degrades when contacted with a nuclease is greater than the rate at which the factor degrades when contacted with the nuclease.

6. A composition in accordance with claim 1, wherein said active agent is a therapeutic agent or a diagnostic agent.

7. A composition in accordance with claim 1, wherein said active agent is selected from the group consisting of an antibacterial agent, an antiviral agent, and antiproliferative agent, an antifungal agent, and immunosuppresive agent, and an analgesic.

8. A composition in accordance with claim 1, wherein the rate at which the scaffold degrades when contacted with an endonuclease is greater than the rate at which the factor degrades when contacted with the endonuclease.

9. A composition in accordance with claim 1, wherein the rate at which the scaffold degrades when contacted with an exonuclease is greater than the rate at which the factor degrades when contacted with the exonuclease.

10. A composition in accordance with claim 1, wherein said scaffold and factor oligonucleotide are at least 80% complementary.

11. A composition in accordance with claim 1, wherein said scaffold and factor oligonucleotide are at least 90% complementary.

12. A composition in accordance with claim 1, wherein said scaffold and factor oligonucleotide are 100% complementary.

13. A composition in accordance with claim 1, comprising a plurality of oligonucleotide factors that can be the same or different.

14. A composition in accordance with claim 13, wherein said oligonucleotide factors are the same.

15. A composition in accordance with claim 13, wherein said oligonucleotide factors are different.

16. A composition in accordance with claim 13, further comprising a plurality of active agents attached to said plurality of oligonucleotide factors, wherein the active agents are the same or different.

17. A composition in accordance with claim 1, wherein: (a) said active agent is covalently attached to said oligonucleotide factor, (b) said scaffold and factor oligonucleotides each comprise from 5 to 100 bases and are at least 90% complementary, and (c) the rate at which the scaffold degrades when contacted with an endonuclease is greater than the rate at which the factor degrades when contacted with the endonuclease.

18. A composition in accordance with claim 1, wherein: (a) said active agent is covalently attached to said oligonucleotide factor, and (b) said scaffold and factor oligonucleotides each comprise from 5 to 100 bases and are at least 90% complementary, and (c) the rate at which the scaffold degrades when contacted with an exonuclease is greater than the rate at which the factor degrades when contacted with the exonuclease.

19. A composition in accordance with claim 1, wherein said oligonucleotide factor has an attached targeting moiety.

20. A composition in accordance with claim 19, wherein said targeting moiety is an antibody or peptide having specificity for a cell-surface receptor.

21. A composition in accordance with claim 1, wherein said oligonucleotide scaffold has an attached targeting moiety.

22. A composition in accordance with claim 21, wherein said targeting moiety is an antibody or peptide having specificity for a cell-surface receptor.

23. A composition in accordance with claim 19, wherein: (a) said targeting moiety is an antibody or peptide having specificity for a cell-surface receptor, (b) said active agent is covalently attached to said oligonucleotide factor, (c) said scaffold and factor oligonucleotides each comprise from 5 to 100 bases and are at least 90% complementary, and (d) the rate at which the scaffold degrades when contacted with an endonuclease is greater than the rate at which the factor degrades when contacted with the endonuclease.

24. A composition in accordance with claim 19, wherein: (a) said targeting moiety is an antibody or peptide having specificity for a cell-surface receptor, (b) said therapeutic agent is covalently attached to said oligonucleotide factor, (c) said scaffold and factor oligonucleotides each comprise from 5 to 100 bases and are at least 90% complementary, and (d) the rate at which the scaffold degrades when contacted with an exonuclease is greater than the rate at which the factor degrades when contacted with the exonuclease.

25. A composition in accordance with claim 21, wherein: (a) said targeting moiety is an antibody or peptide having specificity for a cell-surface receptor, (b) said active agent is covalently attached to said oligonucleotide factor, (c) said scaffold and factor oligonucleotides each comprise from 5 to 100 bases and are at least 90% complementary, and (d) the rate at which the scaffold degrades when contacted with an endonuclease is greater than the rate at which the factor degrades when contacted with the endonuclease.

26. A composition in accordance with claim 21, wherein: (a) said targeting moiety is an antibody or peptide having specificity for a cell-surface receptor, (b) said therapeutic agent is covalently attached to said oligonucleotide factor, (c) said scaffold and factor oligonucleotides each comprise from 5 to 100 bases and are at least 90% complementary, and (d) the rate at which the scaffold degrades when contacted with an exonuclease is greater than the rate at which the factor degrades when contacted with the exonuclease.

27. A method for the controlled release of an active agent in a subject, said method comprising administering to said subject a composition of claim 1.

28. A method for the controlled release of an active agent in a subject, said method comprising administering to said subject a composition of claim 17.

29. A method for the controlled release of an active agent in a subject, said method comprising administering to said subject a composition of claim 18.

30. A method for the delivery of an active agent to a subject, said method comprising contacting said subject with a solid support-bound controlled release composition, said composition comprising: (a) an oligonucleotide scaffold attached to said solid support; (b) an oligonucleotide factor, said scaffold and factor being substantially complementary; and (c) an active agent attached to said oligonucleotide factor; wherein the rate at which the scaffold degrades is greater than the rate at which the factor degrades.

31. A method in accordance with claim 30, wherein said solid support is selected from the group consisting of a gel, an implantable biosensor, and embolization coil, an orthopedic prosthetic, an implantable three dimensional matrix, and a stent.

32. A kit for the preparation of a controlled release active agent composition, said kit comprising: (a) a first container holding an oligonucleotide scaffold; and (b) a second container holding an oligonucleotide factor having an attached active agent; wherein the rate at which the scaffold degrades when contacted with a nuclease is greater than the rate at which the factor degrades when contacted with the nuclease.

33. A method for simultaneously investigating the function of a plurality of genes in a cell, the method comprising: (a) placing a cell culture onto a support having a plurality of known locations, wherein: (i) each known location is attached with a composition comprising a distinct oligonucleotide scaffold and a distinct antisense oligonucleotide factor, (ii) the scaffold and the factor are substantially complementary, (iii) each factor is configured to inhibit the expression of a known gene, and (iv) the rate at which the scaffold degrades when contacted with a nuclease is greater than the rate at which the factor degrades when contacted with the nuclease; (b) contacting the culture with a nuclease; (c) allowing the antisense factors to release from the scaffolds and react with the cells: (d) immunostaining the cells for protein expression; and (e) analyzing the cells in the vicinity of each known location to determine the effect of the antisense factor on the physiology of the cell.

34. A method of detecting the presence of nuclease activity in a test sample, the method comprising: (a) contacting the test sample to a complex, wherein: (i) the complex comprises an oligonucleotide scaffold and an oligonucleotide factor, (ii) the scaffold and the factor are substantially complementary, (iii) the factor is attached with a label, and (v) the rate at which the scaffold degrades when contacted with a nuclease is greater than the rate at which the factor degrades when contacted with the nuclease; (b) allowing the test sample to react with the complex; (c) detecting whether labeled factor is released from the scaffold, thus indicating the presence of nuclease activity in the test sample.

35. A composition in accordance with claim 1, wherein the rate at which the scaffold degrades is at least twice as great as the rate at which the factor degrades.

36. A composition in accordance with claim 1, wherein the rate at which the scaffold degrades is at least ten times as great as the rate at which the factor degrades.

37. A composition in accordance with claim 1, wherein the oligonucleotide scaffold is gel-forming DNA.

38. A composition for the controlled release of a biologically active oligonucleotide factor, said composition comprising: (a) an oligonucleotide scaffold; and (b) an oligonucleotide factor, said scaffold and factor oligonucleotides being substantially complementary; wherein the rate at which the scaffold degrades is greater than the rate at which the factor degrades, and the oligonucleotide factor is biologically active upon release from said oligonucleotide scaffold.

39. A composition in accordance with claim 38, wherein said oligonucleotide factor is an antisense oligonucleotide.

40. A composition in accordance with claim 39, wherein said antisense oligonucleotide inhibits the expression of hepatitus C virus proteins.

41. A composition in accordance with claim 38, wherein said oligonucleotide factor is a gene therapy agent.

42. A composition in accordance with claim 38, wherein said oligonucleotide factor is a ribozyme.

43. A method for transporting and releasing a biologically active oligonucleotide into a cell, said method comprising contacting said cell with a composition of claim 38.

44. A method in accordance with claim 43, wherein said oligonucleotide factor is selected from the group consisting of an antisense oligonucleotide, a gene therapy agent and a ribozyme.