Patent Application
20100015116
Intoxication with biological toxins is a serious health and bioterrorism threat problem. Our inability to effectively treat toxin exposure makes certain toxins particularly dangerous agents for terrorist attacks. Treatment options for individuals after toxin infection are limited. For example, once someone is intoxicated with botulinum neurotoxin, the individual is paralyzed for periods up to 4 to 6 months or longer depending on the toxin serotype because the toxin is slow to degrade. During this time the patient is entirely dependent on ventilatory assistance.
Hence, a need exists for an effective treatment after intoxication by toxin. A further need exists for a composition that can inhibit the toxin but also cause the toxin to be degraded to alleviate symptoms of the toxin infection.
The present invention relates to a recombinant ubiquitin ligase molecule that includes a toxin binding domain that has an affinity for an enzymatically active fragment of one or more toxins or toxin serotypes; and an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated ubiquitination of the toxin enzymatically active fragment. The toxin binding domain, in an embodiment, can be a non-cleavable SNARE polypeptide or fragment thereof that binds to the enzymatically active fragment of a botulinum neurotoxin (BoNT). In particular, the enzymatically active fragments of BoNT include those known in the art or from later discovered serotypes, such as BoNT serotypes A-G. Examples of SNARE polypeptides include a non-cleavable SNAP-25, syntaxin, or synaptobrevin. In another embodiment, wherein the toxin binding domain includes a G protein αs polypeptide or fragment thereof that has affinity to the enzymatically active fragment of a cholera toxin. The toxin binding domain that has an affinity for an enzymatically active fragment of one or more of the following toxins The toxin includes, botulinum neurotoxin (BONT) (serotypes A-G), Clostridia difficile toxins A and B (Tcd A and Tcd B), Clostridium Lethal Toxin, Anthrax Lethal Factor (LF), Ricin, Exotoxin A, Diphtheria toxin, Cholera toxin, Tetanus toxin, Shiga toxin, and any combinations thereof In one aspect, a polypeptide that facilitates E2-mediated ubiquitination is an antibody specific to E2. Another aspect of the invention includes a recombinant ubiquitin ligase having one or more of the following E3 ligase domains: RING, HECT, U-box, RIBRR, F-box domain, DCAF domain, DDS2, HIF-mimetic peptides, IkB-mimetic sequences, BTB domain or combination thereof.
In particular, the recombinant ubiquitin ligase molecule of the present invention includes a non-cleavable SNARE polypeptide or fragment thereof is encoded by a nucleic acid molecule that comprises one or more of the following nucleic acid sequences: a nucleic acid sequence having greater than or equal to about 70% identity with SEQ ID NO: 5; a nucleic acid sequence complement having greater than or equal to about 70% identity with of SEQ ID NO: 5; a nucleic acid molecule having greater than or equal to about 70% identity with a molecule that hybridizes to SEQ ID NO: 5; and a nucleic acid sequence that encodes an amino acid sequence having greater than or equal to about 70% similarity to a sequence set forth in SEQ ID NO: 6. Similarly, the present invention further relates to a recombinant ubiquitin ligase molecule, wherein the non-cleavable SNARE polypeptide or fragment thereof that comprises one or more of the following amino acid sequences: an amino acid sequence encoded by a nucleic acid having greater than or equal to about 70% identity with SEQ ID NO: 5; an amino acid sequence encoded by a complement having greater than or equal to about 70% identity with of SEQ ID NO: 5; an amino acid sequence encoded by a nucleic acid molecule having greater than or equal to about 70% identity with a molecule that hybridizes to SEQ ID NO: 5; and an amino acid sequence having greater than or equal to about 70% similarity to a sequence set forth in SEQ ID NO: 6.
The recombinant ubiquitin ligase molecule of the present invention includes a SNARE polypeptide or fragment that has been mutated to be non-cleavable. In particular, the molecule of the present invention is encoded by a nucleic acid molecule that comprises one or more of the following nucleic acid sequences: a nucleic acid sequence having greater than or equal to about 70% identity with SEQ ID NO: 1 or 3; a nucleic acid sequence complement having greater than or equal to about 70% identity with of SEQ ID NO: 1 or 3; a nucleic acid molecule having greater than or equal to about 70% identity with a molecule that hybridizes to SEQ ID NO: 1 or 3; and a nucleic acid sequence that encodes an amino acid sequence having greater than or equal to about 70% similarity to a sequence set forth in SEQ ID NO: 2 or 4. These nucleic acid molecules include one or more mutations to render the polypeptide non-cleavable. Along the same lines, the present invention relates to a recombinant ubiquitin ligase molecule that comprises one or more of the following amino acid sequences: an amino acid sequence encoded by a nucleic acid having greater than or equal to about 70% identity with SEQ ID NO: 1 or 3; an amino acid sequence encoded by a complement having greater than or equal to about 70% identity with of SEQ ID NO: 1 or 3; an amino acid sequence encoded by a nucleic acid molecule having greater than or equal to about 70% identity with a molecule that hybridizes to SEQ ID NO: 1 or 3; and an amino acid sequence having greater than or equal to about 70% similarity to a sequence set forth in SEQ ID NO: 2 or 4; wherein the nucleic acid molecule comprises one or more mutations to render the polypeptide non-cleavable.
The present invention pertains to a recombinant ubiquitin ligase molecule having a E3 ligase domain that is encoded by a nucleic acid molecule that comprises one or more of the following nucleic acid sequences: a nucleic acid sequence having greater than or equal to about 70% identity with SEQ ID NO: 9; a nucleic acid sequence complement having greater than or equal to about 70% identity with of SEQ ID NO: 9; a nucleic acid molecule having greater than or equal to about 70% identity with a molecule that hybridizes to SEQ ID NO: 9; and a nucleic acid sequence that encodes an amino acid sequence having greater than or equal to about 70% similarity to a sequence set forth in SEQ ID NO: 10. Similarly, the present invention includes a recombinant ubiquitin ligase molecule having a E3 ligase domain that comprises one or more of amino acid sequences: an amino acid sequence encoded by a nucleic acid having greater than or equal to about 70% identity with SEQ ID NO: 9; an amino acid sequence encoded by a complement having greater than or equal to about 70% identity with of SEQ ID NO: 9; an amino acid sequence encoded by a nucleic acid molecule having greater than or equal to about 70% identity with a molecule that hybridizes to SEQ ID NO: 9; and an amino acid sequence having greater than or equal to about 70% similarity to a sequence set forth in SEQ ID NO: 10.
Additionally, the present invention relates to a recombinant ubiquitin ligase molecule that is encoded by a nucleic acid molecule that comprises one or more of the following nucleic acid sequences: a nucleic acid sequence having greater than or equal to about 70% identity with SEQ ID NO: 11 or 13; a nucleic acid sequence complement having greater than or equal to about 70% identity with of SEQ ID NO: 11 or 13; a nucleic acid molecule having greater than or equal to about 70% identity with a molecule that hybridizes to SEQ ID NO: 11 or 13; and a nucleic acid sequence that encodes an amino acid sequence having greater than or equal to about 70% similarity to a sequence set forth in SEQ ID NO: 12 or 14. Also, the present invention includes a recombinant ubiquitin ligase molecule that comprises one or more of the following amino acid sequences: an amino acid sequence encoded by a nucleic acid having greater than or equal to about 70% identity with SEQ ID NO: 11 or 13; an amino acid sequence encoded by a complement having greater than or equal to about 70% identity with of SEQ ID NO: 11 or 13; an amino acid sequence encoded by a nucleic acid molecule having greater than or equal to about 70% identity with a molecule that hybridizes to SEQ ID NO: 11 or 13; and an amino acid sequence having greater than or equal to about 70% similarity to a sequence set forth in SEQ ID NO: 12 or 14.
The present invention pertains to an isolated polypeptide molecule that includes a non-cleavable SNARE polypeptide or fragment thereof that has affinity to the enzymatically active fragment of a BoNT; a BoNT holotoxin, wherein the BoNT holotoxin comprises a deletion in the light chain, a mutation of the heavy chain, or both, wherein the deletion, mutation or both renders BoNT holotoxin atoxic; and an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated degradation of the toxin enzymatically active fragment of a BoNT. The non-cleavable SNARE polypeptide and the BoNT atoxic holotoxin can be fused e.g., the non-cleavable SNARE polypeptide or fragment thereof is fused to the N-terminus of the BoNT atoxic holotoxin. The present invention further relates to fusion proteins that include the isolated polypeptide of the present invention.
In yet another embodiment, the recombinant ubiquitin ligase molecule of the present invention relates to a non-cleavable SNARE polypeptide or fragment thereof that has affinity to the enzymatically active fragment of a BoNT; an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated ubiquitination of the toxin enzymatically active fragment; and a translocation/cellular binding domain. The translocation/cellular binding domain binds to the cellular membrane of a cell and delivers the recombinant ubiquitin ligase into the cell. Examples of the translocation/cellular binding domain include an antennapedia protein, HIV TAT protein, herpes simplex virus VP22 protein, penetratin-derived peptides, kFGF, human β3 integrin, L- and D-arginine oligomers, SCWKn, (LARL)n, HA2; RGD; K1 6RGD oligomer; AlkCWK18, DiCWK18, DipaLytic; Plae, Kplae, MPG peptide, Pep-1, or an atoxic neurotoxin.
Methods of degrading or inhibiting one or more BoNT serotypes that have intoxicated one or more cells are include in the present invention. The steps of the method include contacting an amount of the recombinant ubiquitin ligase of the present invention with the intoxicated cells; wherein the recombinant ubiquitin ligase degrades or inhibits at least one BoNT serotype. An amount of recombinant ubiquitin ligase ranges from e.g., about 1 pM to about 100 mM. The time for contacting the designer ligase of the present invention with intoxicated cells ranges from about 1 minute and about 1 week.
The present invention also relates to methods of treating an individual having one or more cells intoxicated with one or more BoNT serotypes. The steps of the present invention include administering to the individual an amount of recombinant ubiquitin ligase of present invention in a carrier; wherein one or more symptoms associated with BoNT intoxication are reduced or reversed. One or more symptoms associated with BoNT intoxication that are reduced or revised include blurred vision, dry mouth, difficulty swallowing, difficulty speaking, paralysis, muscle weakness; respiratory failure, and decreased nerve conduction. The amount of recombinant ubiquitin ligase administered to the individual ranges from e.g., about 10 ng to about 5 gm. The recombinant ubiquitin ligase can be administered intravenously, parenterally, orally, nasally, by inhalation, by implant, by injection, or by suppository, and can be administered once or periodically. Additionally, the present invention relates to pharmaceutical compositions comprising the recombinant ubiquitin ligase molecule of the present invention and a carrier.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
A description of preferred embodiments of the invention follows.
The present invention relates to a recombinant ubiquitin ligase molecule, also referred to herein as a “designer ubiquitin ligase”. In one aspect, the molecule of the present invention has at least two domains: a toxin binding domain that is specific for the enzymatically active portion of a toxin, referred to herein as the “toxin active fragment”, and an E3 ligase domain that facilitates E2-mediated ubiquitination. See
The present invention relates to a toxin binding domain that has an affinity to or binds to the enzymatically active portion of a toxin. The enzymatically active portion of the toxin is referred to herein as a “toxin active fragment.” The toxin active fragment can be from any toxin known or later discovered or developed. A toxin is a molecule, generally produced by a living cell or organism, which gets into the cell and causes disease or injury. Certain toxins come from animals such as spiders, snakes, pufferfish, scorpions, jellyfish, snails, and bees. Types of toxins include, e.g., neurotoxins (e.g., Botulinum neurotoxin), hemotoxins, necrotoxins, cyanotoxins, plant toxins, and mycotoxins. A neurotoxin is a toxin that affects neurons. Several toxins generally interact with membrane proteins such as ion channels (e.g., sodium, potassium, or calcium channels). A common effect is paralysis, which often sets in very rapidly. Examples of neurotoxins include botulinum neurotoxin (BoNT), tetanospasmin, tetrodotoxin, and conotoxins. Other protein toxins implicated by the invention are Tcd A and B, Clostridium Lethal Toxin, Anthrax Lethal Factor, Ricin, Exotoxin A, Diphtheria toxin, Cholera toxin, Tetanus toxin, Shiga toxin, and a combination thereof.
In an embodiment, BoNT or an enzymatically active portion thereof is used. BoNT is a neurotoxin protein produced by the bacterium, Clostridium botulinum. There are at least seven different BoNT serotypes (A to G), and some of the serotypes have various isotypes (e.g., three isotypes of serotype A have been described). Generally, the BoNT has two chains, a heavy chain (e.g., about 100-kDa) and a light chain (e.g., about 50-kDa ) joined by a disulfide bond. The heavy chain contains a cell binding domain and a translocation domain that allows for the toxin to bind to and enter the cell; this heavy chain portion will be referred to as a cell-binding/translocation domain. The light chain is an enzyme (e.g., a protease) that cleaves a fusion protein (e.g., SNAP-25, syntaxin or synaptobrevin) at a neuromuscular junction, preventing vesicles from anchoring to the membrane to release acetylcholine. By inhibiting acetylcholine release, the toxin interferes with nerve impulses to cause muscle contraction causing the paralysis of muscles, seen in botulism.
The toxin binding domain can bind to a toxin active fragment that has been derivative from a toxin's enzymatically active portion. A “derivative” refers to a molecule with toxin enzymatic activity but contains one or more chemical or functional alterations thereof, as compared to the native enzymatic portion. For instance, the botulinum toxin light chain can be modified so that one or more of its amino acid residues is deleted, modified, replaced, or truncated. For instance, the botulinum toxin light chain can be modified in a way such that, for instance, the modification enhances its properties or decreases undesirable side effects, but that still retains the desired botulinum toxin activity. The botulinum toxin can be derived from any of the botulinum toxin serotypes and/or isoforms produced by the bacterium. Alternatively, the botulinum toxin can be a toxin prepared using recombinant or synthetic chemical techniques (e.g., a recombinant peptide, a fusion protein, or a hybrid neurotoxin, as prepared from subunits or domains of different botulinum toxin serotypes). Additionally, the botulinum toxin active fragment can be in the form of a botulinum toxin precursor, which can itself be non-toxic, for instance a non-toxic zinc protease that becomes toxic on proteolytic cleavage.
“Enzymatically active” portion of the toxin refers to the portion of the toxin that normally gets inside of the cell (e.g., in the endosome or cytosol) and is active. Toxins are often made up of at least two parts, a cell-binding/translocation domain, and an enzymatically active domain. In the BoNT, the enzymatically active domain is often referred to as the “light chain.” However, the enzymatically active domain for other toxins can have other names. For example, with the ricin toxin, the enzymatically active domain is the “A” Chain. The cell-binding domain and translocation domain, referred to as the cell-binding/translocation domain, facilitate binding of the toxin active fragment to the cell membrane and transporting the toxin active fragment across the cellular membrane. For certain toxins like the BoNT, this domain is referred to as the heavy chain. For other toxins, such as ricin, this is referred to as the B Chain.
In an embodiment, enzymatically active refers to a protein that causes the cleavage of one or more proteins in the cell, which in turn causes toxic effects. In the case of certain toxins, the enzymatically active domain cleaves a SNARE (“Soluble NSF Attachment Receptor”) protein. SNARE proteins are a large protein superfamily consisting of several members. The primary role of SNARE proteins is to mediate fusion of cellular transport vesicles with the cell membrane. The core SNARE complex is formed by four a-helices contributed by synaptobrevin, syntaxin and SNAP-25. Different toxins, serotypes of a certain toxin, or cell types will involve cleavage of different SNARE proteins. Tetanospasmin, e.g., is the neurotoxin produced by the vegetative spore of Clostridium tetani and causes tetanus. BoNT A, C, and E cleave SNAP-25, in addition BoNT C cleaves syntaxin 1. BoNT B, D, F, G and tetanus toxin cleave VAMP-2. More than one SNARE protein can be cleaved by a single toxin active fragment.
Botulinum toxin is a zinc-dependent protease. As described herein, enzymatic activity resides generally in the light chain of the molecules. These enzymes cleave SNARE proteins, synaptobrevin 1 and 2, syntaxin and SNAP 25, which form the core of a complex involved in the fusion of transmitter-containing vesicles with the plasma membrane. Prior to fusion, the SNARE proteins in the vesicle and plasma membrane interact forming a complex which contracts with an increase in the intracellular calcium concentration, pulling the vesicle close to the plasma membrane. Interaction between lipids in the two membranes allows the vesicle and nerve terminal active zone to fuse. During this fusion, the contents of the vesicles, mainly neurotransmitters, are released, and the inner surface of the vesicles is exposed to the synaptic cleft. If one of the SNARE proteins is cleaved by a neurotoxin, complex formation cannot occur and fusion is interrupted.
The present invention further involves using a non-cleavable recombinant SNARE protein as the toxin binding domain. Any one of the SNARE proteins can be made non-cleavable using DNA recombinant technology. The non-cleavable SNARE protein can be made non-cleavable with mutations, deletion and/or truncations in the sequence. The sequence is altered so the molecule is non-cleavable by the toxin, but still binds to the light chain or enzymatically active fragment of the toxin. A non-cleavable SNARE endoprotease can bind the toxin active fragment, but will not be cleaved, thereby rending the light chain inactive. Since the non-cleavable SNARE also has an E3-ligase domain, the toxin is ubiquitinated and degraded. Various E3 domains for use in the designer ligase of the present invention are described herein. Additionally, methods for mutating, truncating and deleting nucleic acid sequences are described herein.
As described above, a SNARE endoprotease includes SNAP25A and B.
Another polypeptide that can be used as the toxin binding domain is the G protein αs molecule implicated with the Cholera Toxin. The enzymatically active fragment of the Cholera toxin, the Al fragment of the toxin A subunit enters the cytosol, where it activates the G protein α2 through an ADP-ribosylation reaction. By fusing this protein to an E3 domain, and optionally to a delivery system, the designer ubiquitin ligase of the present invention can bind to the enzymatically active portion of the toxin and facilitate ubiquitination of the cholera toxin.
With respect to the BoNT serotypes, the light chain for each serotype has an amino acid sequence, or is encoded by a nucleic acid sequence. The present invention specifically relates a toxin binding domain that binds to the light chain of any of the BoNT serotypes, as well as any recombinant, mutated, truncated or deleted portions thereof. As such, the toxin active fragment can be the recombinant form of any enzymatically active portion thereof (e.g., the light chain of a BoNT serotype).
The toxin binding domain can be made from recombinant DNA which transcribes the desired amino acid sequence that is specific to the toxin active fragment. The recombinant nucleic acid sequence can be a nucleotide “variant” of a toxin binding domain. A variant is a sequence that differs from the known nucleotide sequence for that molecule in having a truncation, and/or one or more nucleotide deletions, substitutions or additions. Such modifications can be readily introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis as taught, for example, by Adelman et al. (DNA 2:183, 1983). Nucleotide variants can be naturally occurring allelic variants, or non-naturally occurring variants. Variant nucleotide sequences preferably exhibit at least about 70%, more preferably at least about 80% and most preferably at least about 90% homology to the recited sequence. Such variant nucleotide sequences will generally hybridize to the recited nucleotide sequence under stringent conditions. In one embodiment, “stringent conditions” refers to prewashing in a solution of 6×SSC, 0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C. The specific sequences, variants of the toxin binding domain are further described herein.
E3 Ligase Domain That Facilitates E2-mediated Ubiquitination
An embodiment of present invention relates to a designer ubiquitin ligase or recombinant ubiquitin ligase that includes the toxin binding domain, as described herein and an E3 ligase domain. The E3 ligase domain facilitates ubiquitination of the complex. Ubiquitination occurs, in an embodiment, when an E2 enzyme interacts with a specific E3 partner and transfers the ubiquitin to the toxin active fragment.
In particular, an E3 ligase can be a multimeric protein complex or a single chain protein that includes various complexes such as E2 binding domains. Examples of various E3 ligase domains include one or more RING, HECT, U-box, RIBRR, F-box domain, DCAF domain, DDS2, HIF-mimetic peptides, IkB-mimetic sequences, BTB domain, or combination thereof.
These E3 ligase domains facilitate ubiquitination, and when fused with the toxin binding domain that is specific to the enzymatically active portion of the toxin, allows for the degradation of the toxin active fragment. Any E3 ligase domains including E2 binding domains known or later discovered or developed can be used. Recombinant E3 ligase domains can be used. Known E3 ligase-binding domains can be modified to increase its ability to induce ubiquitination and bind to E2. As described herein, proteins and their nucleic acids can be modified and expressed.
“Facilitating ubiquitination” refers to aiding or assisting in the attachment of one or more ubiquitin monomers via E2, which in turn, serves as a recognition site for proteasomal degradation. See
In an embodiment, the present invention relates an E3 ligase domain that is an antibody or antibody fragment that binds E2. The term “antibody fragment” refers to portion of an immunoglobulin having specificity to E2 or a molecule involved with its function. The term, “antibody fragment”, is intended to encompass fragments from both polyclonal and monoclonal antibodies including transgenically produced antibodies, single-chain antibodies (scFvs), recombinant Fabs, heavy-chain-only antibodies, and specifically recombinant shark or camelid single chain antibodies (VHHs). VHHs are also referred to as nanobodies.
Nanobodies are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy-chain antibodies. The Nanobody technology is based on fully functional antibodies from camelids that lack light chains. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). The cloned and isolated VHH domain is a stable polypeptide harboring the antigen-binding capacity of the original heavy-chain antibody. (Ablynx, Ghent, Belgium).
Suitable methods of producing or isolating antibody fragments of the requisite specificity are known in the art and include for example, methods which select recombinant antibody from a library, by PCR.
Functional fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered or single chain antibodies, can also be produced. Functional fragments or portions of the foregoing antibodies include those which are reactive with E2. For example, antibody fragments capable of binding to E2 include, but not limited to, scFvs, Fabs, VHHs, Fv, Fab, Fab′ and F(ab′)2 are encompassed by the invention. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can generate Fab or F(ab′)2 fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab′)2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and hinge region of the heavy chain. Accordingly, the present invention encompasses a polynucleic acid that encodes the an antibody that binds E2.
In an embodiment of the present invention, the designer ubiquitin ligase has a translocation and cell binding domain that binds to the cellular membrane of a cell and delivers the polypeptide molecule into the cell. This domain is also referred to as a “delivery vehicle” and can be used interchangeably. The translocation and cellular binding (TCB) domain, in an embodiment, is fused or otherwise attached, directly or indirectly, to the toxin binding domain, as described herein. The polypeptide of the present invention and further include the E3 ligase domain, also described herein.
In an embodiment, the TCB domain can be the heavy chain, chain “B” or the otherwise cell binding/translocation domain of a toxin. For example, the following domains can be used as the TCB domain: BoNT serotype, or an atoxic Tcd A or B. The heavy chain of a toxin can bind the cellular membrane of the intoxicated cell, and allow the transport of the molecule of the present invention to the inside of the cell.
In yet another embodiment, an atoxic holotoxin can be used to deliver the molecule of the present invention into the cell. A holotoxin is the entire toxin, both the light (e.g., the enzymatically active portion) and heavy (e.g., the TCB portion) chains of the toxin. An atoxic holotoxin refers to a molecule having both chain types, but mutated so that the light chain is no longer enzymatically active. Atoxic holotoxins can be mutated or deleted in any number of ways using methods known in the art. In the BoNT embodiment, serotype A is mutated/truncated to render the light chain enzymatically inactive. In a certain embodiment, portions of the light chain are deleted or truncated to render it inactive. In yet another embodiment, the heavy chain of the toxin can further be mutated, altered, and/or truncated. Methods of altering nucleic acid molecule are further described herein. The use of one type of delivery system that utilizes mutated light chain BoNT holotoxin is described in U.S. Pat. No. 6,203,794.
The composition of the present invention, the TCB domain can be, for example, covalently linked to the toxin binding domain and/or the E3 ligase domain and further can be, for example, a protein, peptide or peptidomimetic. In one embodiment, the composition of the present invention is a chimeric protein, peptide or peptidomimetic in which the delivery agent is operatively fused having e.g., a length of at most 50 or 100 residues.
A variety of TCB domains can be covalently linked to the toxin binding domain and/or E3 ligase domain and include, e.g., an antennapedia protein or active fragment thereof, such as an active fragment having the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 15); an HIV TAT protein or active fragment thereof, such as an active fragment having the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 16); or a herpes simplex virus VP22 protein or active fragment thereof.
In general, in the composition of the present invention, TCB domain can be, for example, covalently linked to the domains described herein, as a protein, peptide or peptidomimetic. In one embodiment, the present invention is a chimeric protein, peptide or peptidomimetic in which the delivery agent is operatively fused to the toxin binding domain and/or E3 ligase domain. Such a composition can be, for example, a peptide or peptidomimetic having a length of at most 50 or 100 residues. Examples of penetratin-derived peptides that are useful as delivery agents include SEQ ID NO: 17 (RQIKIWFQNRRMKWKK), SEQ ID NO:18 (KKWKMRRNQFWIKIQR); SEQ ID NO: 19 (RQIKIWFQNRRMKWKK); SEQ ID NO: 20 (RQIKIWFPNRRMKWKK); SEQ ID NO:21 (RQPKIWFPNRRMPWKK); SEQ ID NO: 22 (RQIKIWFQNMRRKWKK); SEQ ID NO: 23 (RQIRIWFQNRRMRWRR); and SEQ ID NO:24 (RRWRRWWRRWWRRWRR).
In another embodiment, the composition of the present invention includes a delivery agent which is a HIV trans-activator (TAT) protein or an active fragment thereof. Such a delivery agent can include, for example, a sequence identical or similar to residues 47-57 or 47-59 of HIV TAT. As an example, fusion proteins including residues 47-57 of HIV TAT (YGRKKRRQRRR; SEQ ID NO:25) cross the plasma membrane of, for example, human and murine cells in vitro and in vivo; a variety of proteins from 15 to 120 KDa have been shown to retain biological activity when fused to a HIV TAT delivery agent. An HIV TAT delivery agent can be positively charged and can function, for example, in an energy-, receptor-, transporter- and endocytosis-independent manner to deliver a covalently linked toxin binding domain and/or E3 ligase domain to cells intoxicated with a toxin.
Yet another TCB domain for use with the present invention includes a herpes simplex virus VP22 protein or active fragment thereof. In a particular embodiment, the composition of the present invention includes an HSV type 1 (HSV-1) VP22 protein or active fragment thereof. HSV VP22, a nuclear transcription factor, can cross the plasma membrane through non-classical endocytosis and can enter cells independent of GAP junctions and physical contacts. As a fusion with a variety of different proteins, HSV VP22 results in uptake into cells of different types including terminally differentiated cells and can function to deliver a linked toxin binding domain and/or E3 ligase domain.
An example of another delivery agent useful in the present invention corresponds to or is derived from a hydrophobic signal sequence. Such a delivery agent can be, for example, the Kaposi fibroblast growth factor (kFGF) or an active fragment thereof such as AAVALLPAVLLALLAP (SEQ ID NO:26); human β3 integrin or an active fragment thereof; or another hydrophobic delivery agent known in the art.
A delivery agent that can form a portion of the composition of the present invention also can be a synthetic sequence that shares one or more characteristics of a naturally occurring delivery agent such as a protein transduction domain (PTD). Such delivery agents include, but are not limited to, L- and D-arginine oligomers, for example, 9-mers of L- or D-arginine and related peptoids. Such delivery agents further include basic peptides and peptidomimetics; basic α-helical peptides and peptidomimetics; and peptides and peptidomimetics with optimized arginine alignment or optimized a-helical character as compared to a naturally occurring protein transduction domain such as residues 47-57 of HIV TAT. See, for example, WO 99/29721. Additional examples of delivery agents useful in the invention include SCWKn, (LARL)n, HA2; RGD; K1 6RGD, oligomer; AlkCWK18, DiCWK18, DipaLytic; Plae, Kplae and other delivery agents known in the art or developed in the future.
A delivery agent useful in the present invention also can be an agent that enables or enhances cellular uptake of the domains of the composition of the present invention that are associated non-covalently. In one embodiment, such a delivery agent is peptide containing two independent domains: a hydrophobic domain and a hydrophilic domain. In another embodiment, such a delivery agent is an MPG peptide, which is a peptide derived from both the nuclear localization sequence (NLS) of SV40 large T antigen and the fusion peptide domain of HIV-1 gP41. In a further embodiment, such a delivery agent is an MPG peptide having the amino acid sequence GALFLGFLGAAGSTMGAWSQPKSKRKV (SEQ ID NO:27). In yet a further embodiment, such a delivery agent is an amphipathic peptide such as Pep-1. These and related delivery agents that function in the absence of covalent linkage, also referred to as “protein transfection products,” can be used as the delivery system or the “TCB” domain of the present invention. Such peptide delivery agents/TCB domains for use with the composition of the present invention can be prepared by methods known in the art and/or are commercially available; as an example, the Chariot™ product is available from Active Motif (Carlsbad, Calif.).
The present invention relates to inhibiting one or more toxins, toxin active fragments, or toxin serotypes by contacting the composition of the present invention with the toxin. In particular, these methods are applicable in vitro and in vivo.
In vivo, the composition of the present invention is contacted with a cell intoxicated with the toxin active fragment that is the target of the toxin binding domain of the composition. The cell can be intoxicated using methods known in the art. For example, the cell can be intoxicated if the toxin is the holotoxin, or has the enzymatically active portion of the toxin fused with a cell binding/translocation domain. For example, the light chain of the BoNT/A can be delivered to a cell in any number of ways known in the art, and include exposing the enzymatically active portion of the toxin to the cell in concentrations and subjecting the mixture to conditions that allow entry of the toxin active fragment into the cell.
After the cell is intoxicated with the toxin active fragment or toxin, the intoxicated cell is exposed to or comes into contact with the composition of the present invention. The toxin-binding domain binds the toxin active fragment and the E3 ubiquitin ligase domain facilitates polyubiquitination and the subsequent degradation of the toxin. The composition of the present invention is exposed to the intoxicated cell in an amount ranging from about 1 nM to about 100 mM, and for a length of time ranging from about 1 minute and about 1 week.
In vivo, the composition of the present invention is administered to an individual exposed to the toxin. The toxin enters cells of the individual and often causes paralysis or other symptoms depending on the type of toxin. The present invention includes methods of administering one or more designer ubiquitin ligases, described herein, to an individual. The toxin binding domain binds to the toxin active fragment that has intoxicated the cells of the individual. The amount of recombinant ubiquitin ligase of the present invention can be administered to the individual ranges from about 100 ng to about 5 gm.
In one aspect, the present invention embodies targeting multiple toxins or toxin serotypes. This can be accomplished at least in two ways using the compositions of the present invention. The composition of the present invention can include more than one toxin binding domains, each specific to a different portion of the same toxin active fragment or to different toxin active fragments of different toxins. In another embodiment, multiple designer ubiquitin ligases, each to target different areas of one or more toxins are administered. In an embodiment, two, three or more different enzymatically active portions of a toxin or toxin serotypes (e.g., BoNT serotype A, B, C, etc.), can be used as the target for the toxin binding domain. In a case in which a number of serotypes can be involved in causing a disease or condition, such as botulism, multiple enzymatic portions of the toxin serotypes can be targeted. In the case of botulism, since any one of at least seven neurotoxin serotypes could be responsible for botulism, a molecule having with toxin binding domain to one, all, or any combination of the BoNT serotypes is encompassed by the present invention. Alternatively, a pool of designer ligases can be prepared that contain an toxin binding domain that is specific for the enzymatic portion of one or more known serotypes that cause human disease. Botulism is often caused by exposure to a single BoNT serotype, but it is generally difficult to quickly determine which serotype is the cause. Thus, the standard of care in treating botulism includes administration of a number of antibodies to protect against most if not all of the serotypes that cause the disease in human. Hence, to protect against such a disease, an embodiment of the present invention includes having a cocktail of more than one designer ligase so that the ligases bind to several or preferably all of the serotypes that cause botulism. In such a case, the E3-ligase domain, as further described herein, can remain constant among the various designer ligases, whereas in another embodiment, they can differ.
The methods of the present invention include treating an individual infected with one or more toxins. This is accomplished by administering the designer ubiquitin ligase of the present invention to the infected individual. Administration ameliorates or reduces the severity of one or more the symptoms of the disease or condition. The presence, absence, or severity of symptoms can be measured using tests and diagnostic procedures known in the art. Similarly the presence, absence and/or level of the toxin can be measured using methods known in the art. Symptoms or levels of the toxin can be measured at one or more time points (e.g., before, during and after treatment, or any combination thereof) during the course of treatment to determine if the treatment is effective. A decrease or no change in the level of the disease agent, or severity of symptoms associated therewith indicates that treatment is working, and an increase in the level of the toxin, or severity of symptoms indicates that treatment is not working. Symptoms and levels of toxin are measured using methods known in the art.
In another embodiment, a formulation of the present invention can contain one or more of the DNA molecules that encode the designer ubiquitin ligase or portion thereof either present as a mixture or in the form of a DNA fusion molecule, each DNA molecule encoding a polypeptide as described above, such that the polypeptide is generated in situ. The DNA of the present invention can be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). In a preferred embodiment, the DNA can be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, lentivirus, adenovirus, and adeno-associated virus), which can involve the use of a non-pathogenic (defective), replication competent virus. In particular, adeno-associated viral delivery can be used to deliver nucleic acid molecules that encode the designer ubiquitin of the present invention. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA can also be “naked,” as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
The designer ubiquitin ligase of the present invention can be administered in one or more pharmaceutical carriers. The terms “pharmaceutically acceptable carrier” or a “carrier” refer to any generally acceptable excipient or drug delivery device that is relatively inert and non-toxic. The designer ubiquitin ligase of the present invention can be administered with or without a carrier. Exemplary carriers include calcium carbonate, sucrose, dextrose, mannose, albumin, starch, cellulose, silica gel, polyethylene glycol (PEG), dried skim milk, rice flour, magnesium stearate, and the like. Suitable formulations and additional carriers are described in Remington's Pharmaceutical Sciences, (17th Ed., Mack Pub. Co., Easton, Pa.), the teachings of which are incorporated herein by reference in their entirety. The ubiquitin designer ligase of the present invention can be administered systemically or locally (e.g., by injection or diffusion).
Suitable carriers (e.g., pharmaceutical carriers) also include, but are not limited to sterile water, salt solutions (such as Ringer's solution), alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc. Such preparations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active substances, e.g., enzyme inhibitors, to reduce metabolic degradation. A carrier (e.g., a pharmaceutically acceptable carrier) is preferred, but not necessary to administer one or more designer ubiquitin ligases.
The designer ubiquitin ligase of the present invention can be administered intravenously, parenterally, orally, nasally, by inhalation, by implant, by injection, or by suppository. The composition can be administered in a single dose or in more than one dose over a period of time to confer the desired effect.
The actual effective amounts of compositions of the present invention can vary according to the designer ubiquitin ligase being utilized, the particular composition formulated, the mode of administration and the age, weight and condition of the patient, for example. As used herein, an effective amount of the designer ubiquitin ligase of the present invention is an amount which is capable of reducing one or more symptoms of the disease or conditions caused by the toxin. Dosages for a particular patient can be determined by one of ordinary skill in the art using conventional considerations, (e.g. by means of an appropriate, conventional pharmacological protocol).
The administration of the composition of the present invention and other anti-toxin drugs can occur simultaneously or sequentially in time to confer the desired effect.
Systems or kits of the present invention include one or more designer ubiquitin ligase of the present invention, as described herein.
As used herein, the term “recombinant” refers to a molecule that is one that is genetically made using techniques described herein. The present invention relates to a “recombinant” ubiquitin ligase or portions thereof that are engineered genetically. The present invention also pertains to polypeptide molecules that are encoded by nucleic acid sequences, SEQ ID NO: 5, 9, 11, 13, or combinations thereof).
As used herein, the term “polypeptide” encompasses amino acid chains of the designer ubiquitin ligase having any length, partial or full length proteins, wherein the amino acid residues are linked by covalent peptide bonds. Thus, a polypeptide can comprise a portion of the designer ubiquitin ligase or domain thereof, or it can contain additional sequences. Note that terms, “heavy chain” and “light chain”, can be used describe a portion of the antibody fragment being used, but also can refer to the domain of the toxin (e.g., the light chain is the enzymatically active portion, and the heavy chain is the translocation/cell binding domain), depending on the context the term is being used.
Specifically, the present invention relates to recombinant designer ubiquitin ligases having a toxin binding domain such as the non-cleavable SNAP25 domain (SEQ ID NO: 6) and an E3 ligase domain such as the RING domain (SEQ ID NO: 10). Similarly, the present invention also includes a composition that is encoded by a nucleic acid sequence of SEQ ID NO: 5 and 9.
The polypeptides of the present invention referred to herein as “isolated” are polypeptides that are separated away from other proteins and cellular material of their source of origin. The compositions and methods of the present invention also encompass variants of polypeptides and DNA molecules of the present invention. A polypeptide “variant,” as used herein, is a polypeptide that differs from the recited polypeptide only in conservative substitutions and/or modifications, such that the ability of the designer ubiquitin ligase is retained.
The present invention also encompasses proteins and polypeptides, variants thereof, or those having amino acid sequences analogous to the amino acid sequences of binding agents described herein. Such polypeptides are defined herein as analogs (e.g., homologues), or mutants or derivatives. “Analogous” or “homologous” amino acid sequences refer to amino acid sequences with sufficient identity of any one of the amino acid sequences of the present invention so as to possess the biological activity (e.g., the ability to bind to the toxin). For example, an analog polypeptide can be produced with “silent” changes in the amino acid sequence wherein one, or more, amino acid residues differ from the amino acid residues of any one of the sequence, yet still possesses the function or biological activity of the polypeptide. In particular, the present invention relates to homologous polypeptide molecules having at least about 70% (e.g., 75%, 80%, 85%, 90% or 95%) identity or similarity with SEQ ID NO: 6, 10, 12, 14, or combination thereof. Percent “identity” refers to the amount of identical nucleotides or amino acids between two nucleotides or amino acid sequences, respectfully. As used herein, “percent similarity” refers to the amount of similar or conservative amino acids between two amino acid sequences.
Homologous polypeptides can be determined using methods known to those of skill in the art. Initial homology searches can be performed at NCBI against the GenBank, EMBL and SwissProt databases using, for example, the BLAST network service. Altschuler, S. F., et al., J. Mol. Biol., 215:403 (1990), Altschuler, S. F., Nucleic Acids Res., 25:3389-3402 (1998). Computer analysis of nucleotide sequences can be performed using the MOTIFS and the FindPatterns subroutines of the Genetics Computing Group (GCG, version 8.0) software. Protein and/or nucleotide comparisons were performed according to Higgins and Sharp (Higgins, D. G. and Sharp, P. M., Gene, 73:237-244 (1988) e.g., using default parameters).
The present invention, in one embodiment, includes an isolated nucleic acid molecule having a sequence of SEQ ID NO: 5, 9, 11, 13, or combinations thereof. See
As used herein, an “isolated” nucleotide sequence is a sequence that is not flanked by nucleotide sequences which normally (e.g., in nature) flank the gene or nucleotide sequence (e.g., as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in a cDNA or RNA library). Thus, an isolated gene or nucleotide sequence can include a gene or nucleotide sequence which is synthesized chemically or by recombinant means. Nucleic acid constructs contained in a vector are included in the definition of “isolated” as used herein. Also, isolated nucleotide sequences include recombinant nucleic acid molecules and heterologous host cells, as well as partially or substantially or purified nucleic acid molecules in solution. The nucleic acid sequences that encode the toxin binding domain of the present invention include homologous nucleic acid sequences. “Analogous” or “homologous” nucleic acid sequences refer to nucleic acid sequences with sufficient identity of any one of the nucleic acid sequences described herein, such that once encoded into polypeptides, they possess the biological activity of any one of the toxin binding domains described herein. In particular, the present invention is directed to nucleic acid molecules having at least about 70% (e.g., 75%, 80%, 85%, 90% or 95%) identity with SEQ ID NO: SEQ ID NO: 5, 9, 11, 13, or combinations thereof.
Also encompassed by the present invention are nucleic acid sequences, DNA or RNA, which are substantially complementary to the DNA sequences encoding the polypeptides of the present invention, and which specifically hybridize with their DNA sequences under conditions of stringency known to those of skill in the art. As defined herein, substantially complementary means that the nucleic acid need not reflect the exact sequence of the sequences, but must be sufficiently similar in sequence to permit hybridization with nucleic acid sequence under high stringency conditions. For example, non-complementary bases can be interspersed in a nucleotide sequence, or the sequences can be longer or shorter than the nucleic acid sequence, provided that the sequence has a sufficient number of bases complementary to the sequence to allow hybridization therewith. Conditions for stringency are described in e.g., Ausubel, F. M., et al., Current Protocols in Molecular Biology, (Current Protocol, 1994), and Brown, et al., Nature, 366:575 (1993); and further defined in conjunction with certain assays.
Specific hybridization can be detected under high stringency conditions. “Stringency conditions” for hybridization is a term of art which refers to the conditions of temperature and buffer concentration which permit and maintain hybridization of a particular nucleic acid to a second nucleic acid; the first nucleic acid may be perfectly complementary to the second, or the first and second may share some degree of complementarity which is less than perfect. For example, certain high stringency conditions can be used which distinguish perfectly complementary nucleic acids from those of less complementarity. “High stringency conditions” for nucleic acid hybridizations and subsequent washes are explained, e.g., on pages 2.10.1-2.10.16 and pages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, et al., In: Current Protocols in Molecular Biology, John Wiley & Sons, (1998)). The exact conditions which determine the stringency of hybridization depend not only on ionic strength, temperature and the concentration of destabilizing agents such as formamide, but also on factors such as the length of the nucleic acid sequence, base composition, percent mismatch between hybridizing sequences and the frequency of occurrence of subsets of that sequence within other non-identical sequences. Thus, high stringency conditions can be determined empirically.
By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions which will allow a given sequence to hybridize (e.g., selectively) with the most similar sequences in the sample can be determined. Exemplary conditions are described in the art (Krause, M. H., et al., 1991, Methods Enzymol. 200:546-556). Also, low and moderate stringency conditions for washes are described (Ausubel, et al., In: Current Protocols in Molecular Biology, John Wiley & Sons, (1998)). Washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids. Generally, starting from the lowest temperature at which only homologous hybridization occurs, each ° C. by which the final wash temperature is reduced (holding SSC concentration constant) allows an increase by 1% in the maximum extent of mismatching among the sequences that hybridize. Generally, doubling the concentration of SSC results in an increase in Tm of about 17° C. Using these guidelines, the washing temperature can be determined empirically for high stringency, depending on the level of the mismatch sought. In some embodiments, high stringency conditions include those in which nucleic acid with less than a few mismatches does not bind. High stringency conditions, using these guidelines, lie in a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× (e.g., about 2×), and a reaction time range of between about 30 seconds and about 36 hours.
The present invention also provides vectors, plasmids or viruses containing one or more of the nucleic acid molecules having the sequence of SEQ ID NO: SEQ ID NO: 5, 9, 11, 13, or combinations thereof). Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F. M., et al., Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd ED. (1989).
Any polypeptide, or domain and/or portion thereof, described herein, can be substituted with a peptidomimetic. As used herein, the term “peptidomimetic” is used broadly to mean a peptide-like molecule that functions in the same manner as the polypeptides of the present invention. Such peptidomimetics include chemically modified peptides, peptide-like molecules containing non-naturally occurring amino acids, and peptoids, which are peptide-like molecules resulting from oligomeric assembly of N-substituted glycines, and function in a similar way as the toxin active fragment upon which the peptidomimetic is derived (see, for example, Goodman and Ro, Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry and Drug Discovery” Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages 803-861).
A variety of peptidomimetics are known in the art including, for example, peptide-like molecules which contain a constrained amino acid, a non-peptide component that mimics peptide secondary structure, or an amide bond isostere. A peptidomimetic that contains a constrained, non-naturally occurring amino acid can include, for example, an α-methylated amino acid; an α,α-dialkyl-glycine or α-aminocycloalkane carboxylic acid; an Nα-Cα cyclized amino acid; an Nα-methylated amino acid; a β- or γ-amino cycloalkane carboxylic acid; an α,β-unsaturated amino acid; a β,β-dimethyl or β-methyl amino acid; a β-substituted-2,3-methano amino acid; an NCΔ or Cα-Cα cyclized amino acid; or a substituted proline or another amino acid mimetic. In addition, a peptidomimetic which mimics peptide secondary structure can contain, for example, a nonpeptidic β-turn mimic; γ-turn mimic; mimic of β-sheet structure; or mimic of helical structure, each of which is well known in the art. A peptidomimetic also can be a peptide-like molecule which contains, for example, an amide bond isostere such as a retro-inverso modification; reduced amide bond; methylenethioether or methylenesulfoxide bond; methylene ether bond; ethylene bond; thioamide bond; trans-olefin or fluoroolefin bond; 1,5-disubstituted tetrazole ring; ketomethylene or fluoroketomethylene bond or another amide isostere. One skilled in the art understands that these and other peptidomimetics are encompassed within the meaning of the term “peptidomimetic” as used herein.
In
In
To determine the effect of the SNAP25/NC-RING designer ubiquitin ligase on the rate of degradation of the YFP-BoNT/A LC, N18 neuroblastoma cells were transfected with YFP-BoNT/A LC alone, YFP-BoNT/A LC with SNAP25/NC, YFP-BoNT/A LC with SNAP25/NC-RING. The cells were treated by radiolabeling with 35S-methionine for 60 minutes followed by chase for various periods of time using standard pulse-chase procedures 24 hours after transfection. The cells were harvested, lysed and the radiolabeled YFP-BoNT/A LC immunoprecipitated with anti-GFP antibody. The immunoprecipitated YFP-BoNT/A LC was then assessed by autoradiography and the amount of radiolabel in the YFP-BoNT/A LC determined. The rate of degradation of the YFP-BoNT/A LC was determined by graphing amount of radiolabeled YFP-BoNT/A LC vs chase time as shown in
Taking the results illustrated in
The relevant teachings of all the references, patents and/or patent applications cited herein are incorporated herein by reference in their entirety.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application claims the benefit of U.S. Provisional Application No. 61/060,324, filed Jun. 10, 2008. The entire teachings of the above application are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61060324 | Jun 2008 | US |
