Patent Application
20060105378
Described herein are high affinity nucleic acid ligands to tenascin-C. Also described herein are methods for identifying and preparing high affinity nucleic acid ligands to tenascin-C. The method used herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by Exponential enrichment. Further disclosed are high affinity nucleic acid ligands to tenascin-C. Further disclosed are RNA ligands to tenascin-C. Also included are oligonucleotides containing nucleotide derivatives chemically modified at the 2′-positions of the purines and pyrimidines. Additionally disclosed are RNA ligands to tenascin-C containing 2′-F and 2′OMe modifications. The oligonucleotides of the present invention are useful as diagnostic and/or therapeutic agents.
Tenascin-C is a 1.1-1.5 million Da, hexameric glycoprotein that is located primarily in the extracellular matrix. Tenascin-C is expressed during embryogenesis, wound healing, and neoplasia, suggesting a role for this protein in tissue remodeling (Erickson and Bourdon (1989) Ann Rev Cell Biol 5:71-92). Neoplastic processes also involve tissue remodeling, and tenascin-C is over-expressed in many tumor types including carcinomas of the lung, breast, prostate, and colon, astrocytomas, glioblastomas, melanomas, and sarcomas (Soini et al. (1993) Am J Clin Pathol 100(2):145-50; Koukoulis et al. (1991) Hum Pathol 22(7):636-43; Borsi et al. (1992) Int J Cancer 52(5):688-92; Koukoulis et al. (1993) J Submicrosc Cytol Pathol 25(2):285-95; Ibrahim et al. (1993) Hum Pathol 24(9):982-9; Riedl et al. (1998) Dis Colon Rectum 41(1):86-92; Tuominen and Kallioinen (1994) J Cutan Pathol 21(5):424-9; Natali et al. (1990) Int J Cancer 46(4):586-90; Zagzag et al. (1995) Cancer Res 55(4):907-14; Hasegawa et al. (1997) Acta Neuropathol (Berl) 93(5):431-7; Saxon et al. (1997) Pediatr Pathol Lab Med 17(2):259-66; Hasegawa et al. (1995) Hum Pathol 26(8):838-45). In addition, tenascin-C is overexpressed in hyperproliferative skin diseases, e.g. psoriasis (Schalkwijk et al. (1991) Br J Dermatol 124(1): 13-20), and in atherosclerotic lesions (Fukumoto et al. (1998) J Atheroscler Thromb 5(1):29-35; Wallner et al. (1999) Circulation 99(10):1284-9). Radiolabeled antibodies that bind tenascin-C are used for imaging and therapy of tumors in clinical settings (Paganelli et al. (1999) Eur J Nucl Med 26(4):348-57; Paganelli et al. (1994) Eur J Nucl Med 21(4):314-21; Bigner et al. (1998) J Clin Oncol 16(6):2202-12; Merlo et al. (1997) Int J Cancer 71(5):810-6).
Aptamers against tenascin-C have potential utility for cancer diagnosis and therapy, as well as for diagnosis and therapy of atheroslerosis and therapy of psoriasis. Relative to antibodies, aptamers are small (7-20 kDa), clear very rapidly from blood, and are chemically synthesized. Rapid blood clearance is important for in vivo diagnostic imaging, where blood levels are a primary determinant of background that obscures an image. Rapid blood clearance may also be important in therapy, where blood levels may contribute to toxicity. SELEX technology allows rapid aptamer isolation, and chemical synthesis enables facile and site-specific conjugation of aptamers to a variety of inert and bioactive molecules. An aptamer to tenascin-C would therefore be useful for tumor therapy or in vivo or ex vivo diagnostic imaging and/or for delivering a variety of therapeutic agents complexed with the tenascin-C nucleic acid ligand for treatment of disease conditions in which tenascin-C is expressed.
The dogma for many years was that nucleic acids had primarily an informational role. Through a method known as Systematic Evolution of Ligands by EXponential enrichment, termed the SELEX process, it has become clear that nucleic acids have three dimensional structural diversity not unlike proteins. The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by EXponential Enrichment,” now abandoned, U.S. Pat. No. 5,475,096 entitled “Nucleic Acid Ligands,” U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled “Methods for Identifying Nucleic Acid Ligands,” each of which is specifically incorporated by reference herein in its entirety. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands or aptamers, each having a unique sequence, and which have the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets in the SELEX method. The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
It has been recognized by the present inventors that the SELEX method demonstrates that nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, both entitled “Method for Selecting Nucleic Acids on the Basis of Structure,” describe the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sept. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,” now abandoned, U.S. Pat. No. 5,763,177, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S. patent application Ser. No. 09/093,293, filed Jun. 8, 1998, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,” describe a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737, entitled “High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,” describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, which can be non-peptidic, termed Counter-SELEX. U.S. Pat. No. 5,567,588, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,” describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,” that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat. No. 5,580,737, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2′-amino (2′-NH2), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method of Preparation of Known and Novel 2′ Modified Nucleosides by Intramolecular Nucleophilic Displacement,” now abandoned, describes oligonucleotides containing various 2′-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Chimeric SELEX,” and U.S. Pat. No. 5,683,867, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Blended SELEX,” respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in U.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled “Nucleic Acid Ligand Complexes”. Each of the above described patents and applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
The present invention describes a method for isolating nucleic acid ligands that bind to tenascin-C with high specificity. Further described herein are nucleic acid ligands to tenascin-C. Also described herein are high affinity RNA ligands to tenascin-C. Further described are 2′fluoro-modified pyrimidine and 2′OMe-modified purine RNA ligands to tenascin-C. The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by Exponential enrichment. Included herein are the ligands that are shown in Tables 3 and 4 and
Further included in this invention is a method for detecting the presence of a disease that is expressing tenascin-C in a biological tissue that may contain the disease. Still further included in this invention is a method for detecting the presence of a tumor that is expressing tenascin-C in a biological tissue that may contain the tumor. Further included in this invention is a complex for use in in vivo or ex vivo diagnostics. Still further included in this invention is a method for delivering therapeutic agents for the treatment or prophylaxis of diseased tissues that express tenascin-C. Still further included in this invention is a complex for use in delivering therapeutic agents for treatment or prophylaxis of diseased tissues that express tenascin-C.
The central method utilized herein for identifying nucleic acid ligands to tenascin-C is called the SELEX process, an acronym for Systematic Evolution of Ligands by Exponential enrichment and involves (a) contacting the candidate mixture of nucleic acids with tenascin-C (b) partitioning between members of said candidate mixture on the basis of affinity to tenascin-C, and c) amplifying the selected molecules to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to tenascin-C. The invention includes RNA ligands to tenascin-C. This invention further includes the specific RNA ligands to tenascin-C shown in Tables 3 and 4 and
A review of the sequence homologies of the nucleic acid ligands of tenascin-C shown in Tables 3 and 4 and
Further included in this invention is a method for detecting the presence of a disease that is expressing tenascin-C in a biological tissue which may contain the disease by the method of (a) identifying a nucleic acid ligand from a candidate mixture of nucleic acids, the nucleic acid ligand being a ligand of tenascin-C, by the method comprising (i) contacting a candidate mixture of nucleic acids with tenascin-C, wherein nucleic acids having an increased affinity to tenascin-C relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; (ii) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; (iii) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids with relatively higher affinity and specificity for binding to tenascin-C, whereby a nucleic acid ligand of tenascin-C is identified; (b) attaching a marker that can be used in in vivo or ex vivo diagnostics to the nucleic acid ligand identified in step (iii) to form a marker-nucleic acid ligand complex; (c) exposing a tissue which may contain the disease to the marker-nucleic acid ligand complex; and (d) detecting the presence of the marker-nucleic acid ligand in the tissue, whereby a disease expressing tenascin-C is identified.
It is a further object of the present invention to provide a complex for use in in vivo or ex vivo diagnostics comprising one or more tenascin-C nucleic acid ligands and one or more markers. Still further included in this invention is a method for delivering therapeutic agents for the treatment or prophylaxis of disease conditions in which tenascin-C is expressed. Still further included in this invention is a complex for use in delivering therapeutic agents for treatment or prophylaxis of disease conditions in which tenascin-C is expressed.
Various terms are used herein to refer to aspects of the present invention. To aid in the clarification of the description of the components of this invention, the following definitions are provided:
As used herein, “nucleic acid ligand” is a non-naturally occurring nucleic acid having a desirable action on a target. Nucleic acid ligands are often referred to as “aptamers.” The target of the present invention is tenascin-C, hence the term tenascin-C nucleic acid ligand. A desirable action includes, but is not limited to, binding of the target, catalytically changing the target, reacting with the target in a way which modifies/alters the target or the functional activity of the target, covalently attaching to the target as in a suicide inhibitor, facilitating the reaction between the target and another molecule. In the preferred embodiment, the action is specific binding affinity for a target molecule, such target molecule being a three dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein the nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule. Nucleic acid ligands are identified from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a tenascin-C, by the method comprising: a) contacting the candidate mixture with tenascin-C, wherein nucleic acids having an increased affinity to tenascin-C relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids (see U.S. patent application Ser. No. 08/434,425, filed May 3, 1995, now U.S. Pat. No. 5,789,157, which is hereby incorporated herein by reference).
As used herein, “candidate mixture” is a mixture of nucleic acids of differing sequence from which to select a desired ligand. The source of a candidate mixture can be from naturally-occurring nucleic acids or fragments thereof, chemically synthesized nucleic acids, enzymatically synthesized nucleic acids or nucleic acids made by a combination of the foregoing techniques. In a preferred embodiment, each nucleic acid has fixed sequences surrounding a randomized region to facilitate the amplification process.
As used herein, “nucleic acid” means either DNA, RNA, single-stranded or double-stranded, and any chemical modifications thereof. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3′ and 5′ modifications such as capping.
“SELEX” methodology involves the combination of selection of nucleic acid ligands which interact with a target in a desirable manner, for example binding to a protein, with amplification of those selected nucleic acids. Optional iterative cycling of the selection/amplification steps allows selection of one or a small number of nucleic acids which interact most strongly with the target from a pool which contains a very large number of nucleic acids. Cycling of the selection/amplification procedure is continued until a selected goal is achieved. In the present invention, the SELEX methodology is employed to obtain nucleic acid ligands to tenascin-C.
The SELEX methodology is described in the SELEX Patent Applications.
“SELEX target” or “target” means any compound or molecule of interest for which a ligand is desired. A target can be a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc. without limitation. In this application, the SELEX target is tenascin-C.
“Complex” as used herein means the molecular entity formed by the covalent linking of one or more tenascin-C nucleic acid ligands with one or more markers. In certain embodiments of the present invention, the complex is depicted as A-B-Y, wherein A is a marker; B is optional, and comprises a linker; and Y is a tenascin-C nucleic acid ligand.
“Marker” as used herein is a molecular entity or entities that when complexed with the tenascin-C nucleic acid ligand, either directly or through a linker(s) or spacer(s), allows the detection of the complex in an in vivo or ex vivo setting through visual or chemical means. Examples of markers include, but are not limited to radionuclides, including Tc-99m, Re-188, Cu-64, Cu-67, F-18, 125I, 131I, 111In, 32P, 186Re; all fluorophores, including fluorescein, rhodamine, Texas Red; derivatives of the above fluorophores, including Rhodamine-Red-X; magnetic compounds; and biotin.
As used herein, “linker” is a molecular entity that connects two or more molecular entities through covalent bond or non-covalent interactions, and can allow spatial separation of the molecular entities in a manner that preserves the functional properties of one or more of the molecular entities. A linker can also be known as a spacer. Examples of a linker include, but are not limited to, the (CH2CH2O)6 and hexylamine structures shown in
“Therapeutic” as used herein, includes treatment and/or prophylaxis. When used, therapeutic refers to humans and other animals.
“Covalent Bond” is the chemical bond formed by the sharing of electrons.
“Non-covalent interactions” are means by which molecular entities are held together by interactions other than Covalent Bonds including ionic interactions and hydrogen bonds.
In the preferred embodiment, the nucleic acid ligands of the present invention are derived from the SELEX methodology. The SELEX process is described in U.S. patent application Ser. No. 07/536,428, entitled “Systematic Evolution of Ligands by Exponential Enrichment,” now abandoned, U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands” and U.S. Pat. No. 5,270,163, (see also WO 91/19813), entitled “Methods for Identifying Nucleic Acid Ligands.” These applications, each specifically incorporated herein by reference, are collectively called the SELEX Patent Applications.
The SELEX process provides a class of products which are nucleic acid molecules, each having a unique sequence, and each of which has the property of binding specifically to a desired target compound or molecule. Target molecules are preferably proteins, but can also include among others carbohydrates, peptidoglycans and a variety of small molecules. SELEX methodology can also be used to target biological structures, such as cell surfaces or viruses, through specific interaction with a molecule that is an integral part of that biological structure.
In its most basic form, the SELEX process may be defined by the following series of steps:
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, both entitled “Method for Selecting Nucleic Acids on the Basis of Structure,” describe the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sept. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,” now abandoned, U.S. Pat. No. 5,763,177, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S. patent application Ser. No. 09/093,293, filed Jun. 8 1998, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,” all describe a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737, entitled “High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,” describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. Pat. No. 5,567,588, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Solution SELEX,” describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. Pat. No. 5,496,938, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” describes methods for obtaining improved nucleic acid ligands after SELEX has been performed. U.S. Pat. No. 5,705,337 entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chemi-SELEX,” describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,” that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat. No. 5,637,459, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2′-amino (2′-NH2), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method of Preparation of Known and Novel 2′ Modified Nucleosides by Intramolecular Nucleophilic Displacement,” describes oligonucleotides containing various 2′-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX,” and U.S. Pat. No. 5,683,867, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX,” respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
In U.S. Pat. No. 5,496,938 methods are described for obtaining improved Nucleic Acid Ligands after the SELEX process has been performed. This patent, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” is specifically incorporated herein by reference.
U.S. patent application Ser. No. 08/434,425, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Tissue SELEX,” filed May 3, 1995, now U.S. Pat. No. 5,789,157, describes methods for identifying a nucleic acid ligands to a macromolecular component of a tissue, including cancer cells, and the nucleic acid ligands so identified. This patent is specifically incorporated herein by reference.
One potential problem encountered in the diagnostic or therapeutic use of nucleic acids is that oligonucleotides in their phosphodiester form may be quickly degraded in body fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases before the desired effect is manifest. Certain chemical modifications of the nucleic acid ligand can be made to increase the in vivo stability of the nucleic acid ligand or to enhance or to mediate the delivery of the nucleic acid ligand. See, e.g., U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, now abandoned, and U.S. Pat. No. 5,660,985, both entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,” which is specifically incorporated herein by reference. Modifications of the nucleic acid ligands contemplated in this invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3′ and 5′ modifications such as capping. In preferred embodiments of the instant invention, the nucleic acid ligands are RNA molecules that are 2′-fluoro (2′-F) modified on the sugar moiety of pyrimidine residues.
The modifications can be pre- or post-SELEX process modifications. Pre-SELEX process modifications yield nucleic acid ligands with both specificity for their SELEX target and improved in vivo stability. Post-SELEX process modifications made to 2′-OH nucleic acid ligands can result in improved in vivo stability without adversely affecting the binding capacity of the nucleic acid ligand.
Other modifications are known to one of ordinary skill in the art. Such modifications may be made post-SELEX process (modification of previously identified unmodified ligands) or by incorporation into the SELEX process.
The nucleic acid ligands of the invention are prepared through the SELEX methodology that is outlined above and thoroughly enabled in the SELEX applications incorporated herein by reference in their entirety.
The tenascin-C aptamers of the invention bind to the heparin binding site of the tenascin-C COOH terminus.
In certain embodiments of the present invention, the nucleic acid ligands to tenascin-C described herein are useful for diagnostic purposes and can be used to image pathological conditions (such as human tumor imaging). In addition to diagnosis, the tenascin-C nucleic acid ligands are useful in the prognosis and monitoring of disease conditions in which tenascin-C is expressed.
Diagnostic agents need only be able to allow the user to identify the presence of a given target at a particular locale or concentration. Simply the ability to form binding pairs with the target may be sufficient to trigger a positive signal for diagnostic purposes. Those skilled in the art would be able to adapt any tenascin-C nucleic acid ligand by procedures known in the art to incorporate a marker in order to track the presence of the nucleic acid ligand. Such a marker could be used in a number of diagnostic procedures, such as detection of primary and metastatic tumors and athersclerotic lesions. The labeling markers exemplified herein are technetium-99m and 111In; however, other markers such as additional radionuclides, magnetic compounds, fluorophores, biotin, and the like can be conjugated to the tenascin-C nucleic acid ligand for imaging in an in vivo or ex vivo setting disease conditions in which tenascin-C is expressed (e.g., cancer, atherosclerosis, and psoriasis). The marker may be covalently bound to a variety of positions on the tenascin-C nucleic acid ligand, such as to an exocyclic amino group on the base, the 5-position of a pyrimidine nucleotide, the 8-position of a purine nucleotide, the hydroxyl group of the phosphate, or a hydroxyl group or other group at the 5′ or 3′ terminus of the tenascin-C nucleic acid ligand. In embodiments where the marker is technetium-99m of 111In, preferably it is bonded to the 5′ or 3′ hydroxyl of the phosphate group thereof or to the 5 position of a modified pyrimidine. In the most preferred embodiment, the marker is bonded to the 5′ hydroxyl of the phosphate group of the nucleic acid ligand with or without a linker. In another embodiment, the marker is conjugated to the nucleic acid ligand by incorporating a pyrimidine containing a primary amine at the 5 position, and use of the amine for conjugation to the marker. Attachment of the marker can be done directly or with the utilization of a linker. In the embodiment where technetium-99m or 111In is used as the marker, the preferred linker is a hexylamine linker as shown in
In other embodiments, the tenascin-C nucleic acid ligands are useful for the delivery of therapeutic compounds (including, but not limited to, cytotoxic compounds, immune enhancing substances and therapeutic radionuclides) to tissues or organs expressing tenascin-C. Disease conditions in which tenascin-C may be expressed include, but are not limited to, cancer, atherosclerosis, and psoriasis. Those skilled in the art would be able to adapt any tenascin-C nucleic acid ligand by procedures known in the art to incorporate a therapeutic compound in a complex. The therapeutic compound may be covalently bound to a variety of positions on the tenascin-C nucleic acid ligand, such as to an exocyclic amino group on the base, the 5-position of a pyrimidine nucleotide, the 8-position of a purine nucleotide, the hydroxyl group of the phosphate, or a hydroxyl group or other group at the 5′ or 3′ terminus of the tenascin-C nucleic acid ligand. In the preferred embodiment, the therapeutic agent is bonded to the 5′ amine of the nucleic acid ligand. Attachment of the therapeutic agent can be done directly or with the utilization of a linker. In embodiments in which cancer is the targeted disease, 5-fluorodeoxyuracil or other nucleotide analogs known to be active against tumors can be incorporated internally into existing U's within the tenascin-C nucleic acid ligand or can be added internally or conjugated to either terminus either directly or through a linker. In addition, both pyrimidine analogues 2′2′-difluorocytidine and purine analogues (deoxycoformycin) can be incorporated. In addition, U.S. application Ser. No. 08/993,765, filed Dec. 18, 1997, incorporated herein by reference in its entirety, describes, inter alia, nucleotide-based prodrugs comprising nucleic acid ligands directed to a tumor, for example tenascin-C, for precisely localizing chemoradiosensitizers, and radiosensitizers and radionuclides and other radiotherapeutic agents to the tumor.
It is also contemplated that both the marker and therapeutic agent may be associated with the tenascin-C nucleic acid ligand, such that detection of the disease condition and delivery of the therapeutic agent is accomplished together in one aptamer or as a mixture of two or more different modified versions of the same aptamer. It is also contemplated that either or both the marker and/or the therapeutic agent may be associated with a non-immunogenic, high molecular weight compound or lipophilic compound, such as a liposome. Methods for conjugating nucleic acid ligands with lipophilic compounds or non-immunogenic compounds in a diagnostic or therapeutic complex are described in U.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled “Nucleic Acid Ligand Complexes,” which is incorporated herein in its entirety.
The therapeutic or diagnostic compositions described herein may be administered parenterally by injection (e.g., intravenous, subcutaneous, intradermal, intralesional), although other effective administration forms, such as intraarticular injection, inhalant mists, orally active formulations, transdermal iontophoresis or suppositories, are also envisioned. They may also be applied locally by direct injection, can be released from devices, such as implanted stents or catheters, or delivered directly to the site by an infusion pump. One preferred carrier is physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers may also be used. In one embodiment, it is envisioned that the carrier and the tenascin-C nucleic acid ligand complexed with a therapeutic compound constitute a physiologically-compatible, slow release formulation. The primary solvent in such a carrier may be either aqueous or non-aqueous in nature. In addition, the carrier may contain other pharmacologically acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation. Similarly, the carrier may contain still other pharmacologically-acceptable excipients for modifying or maintaining the stability, rate of dissolution, release, or absorption of the tenascin-C nucleic acid ligand. Such excipients are those substances usually and customarily employed to formulate dosages for parental administration in either unit dose or multi-dose form.
Once the therapeutic or diagnostic composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored either in ready to use form or requiring reconstitution immediately prior to administration. The manner of administering formulations containing tenascin-C nucleic acid ligands for systemic delivery may be via subcutaneous, intramuscular, intravenous, intraarterial, intranasal or vaginal or rectal suppository.
The following examples are provided to explain and illustrate the present invention and are not to be taken as limiting of the invention. Example 1 describes the materials and experimental procedures used in Example 2 for the generation of RNA ligands to tenascin-C. Example 2 describes the RNA ligands to tenascin-C and the predicted secondary structure of a selected nucleic acid ligand. Example 3 describes the determination of minimal size necessary for high affinity binding of a selected nucleic acid ligand, and substitution of 2′-OH purines with 2′-OMe purines. Example 4 describes the biodistribution of Tc-99m labeled tenascin-C nucleic acid ligands in tumor-bearing mice. Example 5 describes the use of a fluorescently labeled tenascin-C nucleic acid ligand to localize tenascin-c within tumor tissue. Example 6 describes detection of tumors in vivo by Aptamer TTA1 (also known as GS7641). Example 7 describes alternative labeling using 111In. Example 8 describes detection of tumors in vivo by Aptamer TTA1. The aptamer was administered to a female patient with recurrent breast cancer. The results are set forth in
The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.
Materials and Methods
Tenascin-C was purchased from Chemicon (Temecula, Calif.). Single-stranded DNA-primers and templates were synthesized by Operon Technologies Inc. (Alameda, Calif.).
The SELEX-process has been described in detail in the SELEX Patent Applications. In brief, double-stranded transcription templates were prepared by Klenow fragment extension of 40N7a ssDNA:
using the 5N7 primer:
which contains the T7 polymerase promoter (underlined). RNA was prepared with T7 RNA polymerase as described previously in Fitzwater and Polisky (1996) Methods Enzymol. 267: 275-301, incorporated herein by reference in its entirety. All transcription reactions were performed in the presence of pyrimidine nucleotides that were 2′-fluoro (2′-F) modified on the sugar moiety. This substitution confers enhanced resistance to ribonucleases that utilize the 2′-hydroxyl moiety for cleavage of the phosphodiester bond. Specifically, each transcription mixture contained 3.3 mM 2′-F UTP and 3.3 mM 2′-F CTP along with 1 mM GTP and ATP. The initial randomized RNA library thus produced comprised 3×1014 molecules. The affinities of individual ligands for tenascin-C were determined by standard methods using nitrocellulose filter partitioning (Tuerk and Gold (1990) Science 249(4968):505-10).
For each round of SELEX, Lumino plates (Labsystems, Needham Heights, Mass.) were coated for 2 hours at room temperature with 200 μl Dulbecco's PBS containing tenascin-C concentrations as shown in Table 1. After coating, wells were blocked using HBSMC+ buffer [20 mM Hepes, pH 7.4, 137 mM NaCl, 1 mM CaCl2, 1 mM MgCl2 and 1 g/liter human serum albumin (Sigma, fraction V) for rounds 1 to 6 while for rounds 7 and 8 wells were blocked HBSMC+ buffer containing 1 g/liter casein (I-block; Tropix). Binding and wash buffer consisted of HBSMC+ buffer containing 0.05% Tween 20. For each SELEX round, RNA was diluted into 100 μl of binding buffer and allowed to incubate for 2 hours at 37° C. in the protein coated wells that were pre-washed with binding buffer. After binding, six washes of 200 μl each were performed. Following the wash step, the dry well was placed on top of a 95° C. heat block for 5 minutes. Standard AMV reverse transcriptase reactions (50 μl) were performed at 48° C. directly in the well and the reaction products utilized for standard PCR and transcription reactions. Two synthetic primers 5N7 (see above) and 3N7a: 5′-TCGCGCGAGTCGTCTG-3′ (SEQ ID NO:3) were used for these template amplification and reverse transcription steps.
For cell SELEX, U251 human glioblastoma cells (Hum. Hered. (1971) 21:238) were grown to confluence in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum (GIBCO BRL, Gaithersburg, Md.) on six-well tissue culture plates (Becton Dickinson Labware, Lincoln Park, N.J.) and washed three times using Dulbecco's PBS supplemented with CaCl2 (DPBS, GIBCO BRL) buffer. RNA labeled internally by transcription (Fitzwater (1996) supra) was incubated with the cells at 37° C. for one hour. The labeled RNA was then removed, and the cells were washed six times for ten minutes each at 37° C. with DPBS. DPBS containing 5 mM EDTA was then added and incubated with the cells for 30 minutes to elute bound RNAs that remained after the washing steps. This RNA was quantitated by a standard liquid scintillation counting protocol and amplified using RT-PCR.
Binding assays for the U251 cells. Internally labeled RNA was incubated at increasing concentrations with confluent U251 cells in six-well tissue culture plates (Becton Dickinson Labware, Lincoln Park, N.J.) at 37° C. for 60 min. Unbound RNA was washed away using three 10 minute washes with DPBS+CaCl2 at 37° C., and bound RNA was collected by disrupting the cells using Trizol (Gibco BRL, Gaithersburg, Md.). Bound RNA was quantitated by liquid scintillation counting.
Cloning and Sequencing. Amplified affinity enriched oligonucleotide pools were purified on an 8% polyacrylamide gel, reverse transcribed into ssDNA and the DNA amplified by the polymerase chain reaction (PCR) using primers containing BamH1 and HindIII restriction endonuclease sites. PCR fragments were cloned, plasmids prepared and sequence analyses performed according to standard techniques (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. 3 vols., Cold Spring Harbor Laboratory Press, Cold Spring Harbor).
Nucleic Acid Ligands to U251 cells were obtained by the SELEX process and are described in U.S. patent application Ser. No. 08/434,425, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Tissue SELEX,” filed May 3, 1995, now U.S. Pat. No. 5,789,157. Subsequently it was determined that the ligands that were obtained were tenascin-C nucleic acid ligands.
To obtain oligonucleotide ligands against human tenascin-C, eight rounds of SELEX were performed using the randomized nucleotide library as described above in Materials and Methods. RNA and protein input into each round is shown in Table 1. After 8 rounds of SELEX, the affinity of the oligonucleotide pool for tenascin-C was 10 nM, and this affinity did not increase with additional SELEX rounds.
To obtain ligands to U251 glioblastoma cells, nine rounds of SELEX were performed using the randomized nucleotide library. After nine rounds of binding to U251 cells and EDTA elution, rounds 3, 5 and 9 were tested for their ability to bind to U251 cells.
The E9 pool (nine rounds of binding and EDTA elution from U251 cells) was then used as a starting point for a SELEX against purified tenascin-C. Two rounds of SELEX using purified tenascin-C were performed as described above. Input protein and RNA concentrations for two rounds of SELEX (E9P1 and E9P2) are described in Table 2.
In summary, three different SELEX experiments were performed: an experiment using purified tenascin-C as the target, an experiment using U251 glioblastoma cells as the target, and an experiment in which the SELEX pool from the U251 glioblastoma cells was used to initiate a SELEX experiment using purified tenascin-C as the target.
All three SELEX experiments were analyzed by cloning and sequencing ligands from round 8 of the purified tenascin-C SELEX (“TN” sequences), from round 9 of the U251 cell SELEX (“E9” sequences), and from round 2 of the U251/tenascin-C hybrid SELEX (“E9P2” sequences). The sequences of 34 unique clones are shown in Table 3, and are divided into two major groups: tenascin-C ligands (“TN” and “E9P2” sequences) and U251 cell ligands (“E9” ligands). Among the tenascin-C ligands, the majority of the clones (65 total) represent one of two distinct sequence classes designated Family I and Family II (
Examination of the variable region of the 12 clones in Family I revealed 7 unique sequences that are related through the consensus sequence GACNYUUCCNGCYAC (SEQ ID NO:12). Examination of the variable region of the 18 clones in Family II revealed sequences that share a consensus sequence CGUCGCC (Table 3). The E9 sequences could be grouped into a related set by virtue of conserved GAY and CAU sequences within the variable regions. The remaining sequences did not appear related to other sequences and were classified as orphans. Three sequences predominate, with E9P2-1, E9P2-2, and TN9 represented 14,16, and 10 times respectively. In the “Orphan” category, one sequence, TN18, was represented twice. Overall, these data represent a highly enriched sequence pool.
Most individuals displayed low nanomolar dissociation constants, with the three most prevalent sequences, TN9 and E9P2-1 and -2, having the highest affinities at 5 nM, 2 nM, and 8 nM. (Table 3). These results indicate that the U251 cell SELEX is a repository for aptamers against tenascin-C, and that only two rounds of SELEX were required to isolate the tenascin-specific ligands from the cell SELEX pool. Oligonucleotide ligands against other proteins can be similarly isolated from the E9 pool using purified protein targets.
Oligonucleotide synthesis procedures were standard for those skilled in the art (Green et al. (1995) Chem Biol 2(10):683-95). 2′-fluoro pyrimidine phosphoramidite monomers were obtained from JBL Scientific (San Luis Obispo, Calif.); 2′-OMe purine, 2′-OH purine, hexylamine, and (CH2CH2O)6 monomers, along with the dT polystyrene solid support, were obtained from Glen Research (Sterling, Va.). Aptamer affinities were determined using nitrocellulose filter partitioning (Green et al., supra).
TN9 was chosen for further analysis based on its high affinity for tenascin-C. We first searched for a minimal sequence necessary for high affinity binding. Using standard techniques (Green et al, supra), it was discovered that nucleotides 3′ of nucleotide 55 were required for binding to tenascin-C, while no nucleotides could be removed from the 5′ end without loss of affinity. To further decrease the TN9's length from 55 nucleotides and retain high affinity binding, we then attempted to define internal deletions of TN9. The first 55 nucleotides of TN9, along with the first 55 nucleotides of related family II ligands TN7, TN21, and TN41, were input into a computer algorithm to determine possible RNA secondary structure foldings (mfold 3.0, M. Zuker, D. H. Mathews & D. H. Turner. Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide. In: RNA Biochemistry and Biotechnology, J. Barciszewski & B. F. C. Clark, eds., NATO ASI Series, Kluwer Academic Publishers, (1999) ). Among many potential RNA foldings predicted by the algorithm, a structure common to each oligonucleotide was found. This structure, represented by oligonucleotide TTA1 in
To increase protection against nuclease activity, purine positions that could be substituted with the corresponding 2′-OMe purines were located. The oligonucleotide was arbitrarily divided into five sectors and all purines within each sector were substituted by the corresponding 2′-OMe purine nucleotide, a total of five oligonucleotides (Table 4, Phase I syntheses). The affinity of each oligonucleotide for tenascin-C was determined, and it was found that all purines within sectors 1,3 and 5 could be substituted without appreciable loss in affinity. Within sectors 2 and 4, individual purines were then substituted with 2′-OMe purines and the effect of affinity was measured (Table 4, Phase III syntheses). From these experiments, it was deduced that substitution of nucleotides G9, G28, G31, and G34, with 2′-OMe G causes loss in affinity for tenascin-C. Therefore these nucleotides remain as 2′-OH purines in the aptamer TTA1.
The aptamer TTA1 (Table 4) was then synthesized with the (CH2CH2O)6 (Spacer 18) linker, a 3′-3′ dT cap for exonuclease protection, a 5′ hexylamine (Table 4), and all purines as 2′-OMe except the 5 Gs indicated in Table 4. A non-binding control aptamer, TTA1.NB, was generated by deleting 5 nucleotides at the 3′ end to produce TTA1.NB. TTA1 binds to tenascin-C with an equilibrium dissociation constant (Kd) of 5 nM, while TTA1.NB has a Kd of>5 μM for tenascin-C.
Nucleotides 10-26 can be replaced by a non-nucleotide ethylene glycol linker. It is therefore likely that TTA1 can be synthesized in two separate pieces, where a break is introduced at the position of the ethylene glycol linker and new 5′ and 3′ ends are introduced. Subsequent to synthesis, the two molecules will incubated together to allow hybrid formation. This method allows introduction of additional amine groups as well as nucleotides at the new 5′ and 3′ ends. The new functionalities could be used for bioconjugation. In addition, two-piece synthesis results in increased chemical synthetic yield due to shortening the length of the molecules.
Aptamer biodistribution was tested by conjugating a Tc-99m chelator (Hi15; Hilger et al. (1998) Tet Lett 39:9403-9406) to the 5′ end of the oligonucleotide as shown in
For biodistribution experiments, U251 xenograft tumors were prepared as follows: U251 cells were cultured in Dulbeccos' Modified Eagle's Medium supplemented with 10% v/v fetal calf serum (Gibco BRL, Gaithersburg, Md.). Athymic mice (Harlan Sprague Dawley, Indianapolis, Ind.) were injected subcutaneously with 1×106 U251 cells. When the tumors reached a size of 200-300 mg (1-2 weeks), Tc-99m labeled aptamer was injected intravenously at 3.25 mg/kg. At indicated times, animals were anesthetized using isoflurane (Fort Dodge Animal Health, Fort Dodge, Iowa), blood was collected by cardiac puncture, and the animal was sacrificed and tissues were harvested. Tc-99m levels were counted using a gamma counter (Wallac Oy, Turku, Finland). Aptamer uptake into tissues was measured as the % of injected dose per gram of tissue (% ID/g).
Images of mice were obtained using a gamma camera. Mice were placed onto the camera (Siemens, LEM+) under anesthesia (isoflurane). Data were collected (30 sec to 10 minutes) and analyzed using Nuclear MAC software version 3.22.2 (Scientific Imaging, Calif.) on a Power MAC G3 (Apple Computer, Calif.).
Biodistribution experiments, Table 5, indicated rapid and specific uptake of the aptamer into tumor tissue; the non-binding aptamer does not remain in the tumor. Blood levels of Tc-99m also cleared rapidly. After three hours, Tc-99m levels brought into the tumor using Hi15-TTA1 had a very long half life (>18 hrs). This indicates that once the aptamer penetrates the tumor, the radiolabel carried with it remains in the tumor for long periods of time. Such data indicate that cytotoxic agents, including radionuclides and non-radioactive agents, conjugated to the aptamer will also remain in the tumor with long half lives.
Tc-99m radioactivity also appears in other tissues, notably the small and large intestines. The hepatobiliary clearance pattern seen here can be readily altered by those skilled in the art, for example by altering the hydrophilicity of the Tc-99m chelator, changing the chelator, or changing the radiometal/chelator pair altogether.
Whole animal images were obtained using Tc-99m labeled Hi15-TTA1 and at 3 hours post-injection. Images obtained from mice injected with Hi15-TTA1, but not from mice injected with Hi15-TTA1.NB, clearly show the tumor (
Materials and Methods
TTA1 and TTA1.NB were synthesized as described above. Succinimdyl Rhodamine-Red-X (Molecular Probes, Eugene, Oreg.) was conjugated to the 5′ amine of the aptamers as described above for H15-NHS conjugation. The Rhodamine-Red-X-conjugated aptamers, TTA1-Red and TTA1.NB-Red, were purified by reversed phase HPLC. U251 cell culture and tumor growth in nude mice were as described above. Five nmol of TTA1-Red or TTA1.NB-Red were injected intravenously into nude mice and at the desired time the animal was placed under anesthesia, perfused with 0.9% NaCl, and sacrificed. The tumor was excised and placed in formalin. After 24 hr in formalin, 10 μM sections were cut and Rhodamine-Red-X was detected using a fluorescence microscope (Eclipse E800, Nikon, Japan).
Results: TTA1-Red has identical affinity for tenascin-C as the unconjugated parent aptamer, TTA1, at 5 nM. We compared tumor fluorescence levels of TTA1-Red and TTA1.NB-Red 10 min post-injection. The binding aptamer, TTA1-Red, strongly stains the tumor but not adjacent tissue (
Aptamer labeling, biodistribution, and nude mouse tumor xenografts were performed as described in Example 4.
Many human tumor types are known to express tenascin-C. To assess the ability of TTA1/GS7641 to target tumor types in addition to glioblastomas, human tumor cell lines were grown as tumors in nude mice. Tumor tissue was tested for expression of human tenascin-C, and those tumors expressing human tenascin-C were tested for aptamer uptake.
Tumor xenograft and biodistribution studies were performed as described in Example 4. To couple DTPA and DOTA to TTA1/GS7641, the cyclic anhydride of each was incubated with the amine-containing TTA1/GS7641 under neutral pH conditions using standard methods. The structures of the DTPA and conjugates are shown in
The biodistribution of In-11 labeled aptamer is markedly different from the Tc-labeled formulation described in Example 4.
TTA1 Aptamer was administered to a female patient with recurrent breast cancer. The thoracic region of the patient was then examined by CT (computed tomography) as well as by SPECT. The results are set forth in
E9P2-4 (5)
E9P2-1 (14)
E9P2-2 (16)
GCGUCGCCG
E9P2-17
E9P2-48
E9P2-14
E9P2-17
E9P2-22
E9P2-40
E9P2-41
E9-8 (3)
E9-15
E9-6
E9-5 (2)
E9-3 (6)
E9-9
This application is a continuation of U.S. patent application Ser. No. 10/031,193, filed Jan. 31, 2002, entitled “Tenascin-C Nucleic Acid Ligands,” currently pending, which application is a §371 National Phase of International Patent Application Serial No. PCT/US00/02167, filed Jan. 28, 2000, entitled “Tenascin-C Nucleic Acid Ligands,” which application is a continuation in part of U.S. patent application Ser. No. 09/364,902, filed Jul. 29, 1999, entitled “Tenascin-C Nucleic Acid Ligands,” now U.S. Pat. No. 6,232,071. Each of these references is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10031193 | Jan 2002 | US |
| Child | 11300662 | Dec 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09364902 | Jul 1999 | US |
| Child | 10031193 | Jan 2002 | US |
