# Patent

## Title
Nanocarriers and their processing for diagnostics and therapeutics

## Abstract

The compositions and methods of the invention provide compositions and methods for preferential targeting of tissues to delivery therapeutic or diagnostic agents. For example, such compounds are useful in the treatment of joint disorders those affecting articulating joints, e.g., injury-induced osteoarthritis as well as autoimmune diseases affecting joint tissue such as rheumatoid arthritis.

## Background
RELATED APPLICATIONS
This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62/113,335, filed Feb. 6, 2015 and Provisional Application No. 61/953,495, filed Mar. 14, 2014, which are incorporated herein by reference in their entirety.


STATEMENT OF GOVERNMENT INTERESTS
This invention was made with government support under 1P20RR024484 awarded by the National Institute of Health (NIH) and National Center for Research Resources (NCRR) and P20GM104937 awarded by the NIH and National Institute of General Medical Sciences (NIGMS). The Government has certain rights in the invention.


FIELD OF THE INVENTION
This invention relates to nanoparticles for delivering agents into cells or bodily tissues.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
The contents of the text file named “21486-62201WO.ST25.txt”, which was created on Apr. 9, 2015 and is 351 KB in size, are thereby incorporated by reference in their entirety.
BACKGROUND
Although progress in drug delivery using nanotechnology has been documented, several challenges remain, particularly with regard to tissue targeting and toxicity. Current delivery systems suffer from significant hindrances such as low targeting efficiency. A major reason for these drawbacks is that tissues have extracellular matrix.
SUMMARY OF THE INVENTION
The compositions and methods of the invention provide a solution to long standing challenges in selective delivery of agents using nanotechnology. Accordingly, the invention features compounds, assemblies of such compounds, a system, or method for selective drug delivery to any bodily tissue (including those that include extracellular matrix tissue) comprising a nanoparticle. Nanoparticles such as rosette nanopieces, lipid nanoparticles, and polymeric nanoparticles composition comprise a cargo compound, wherein a positively-charged nanoparticle and cargo complex composition with net positive charge at pH 7-7.5 localizes or penetrates a negatively-charged tissue or wherein a negatively-charged (or weakly positively-charged) nanoparticle and cargo complex composition with net negative (or weak positive) charge at pH 7-7.5 localizes to or penetrates a positively-charged tissue. “Negatively charged” means zeta-potential of equal or smaller than 0 mV (which is minus “−” mV). “Positively charged” means zeta-potential of equal or larger than 0 mV (which is plus “+” mV). “Weakly positive” means zeta potential of 0 mV to +30 mV. The nanoparticle is tuned to preferentially localize to and deliver its cargo to a target bodily tissue. For example, a relatively negatively charged nanoparticle is used to preferentially localize to, accumulate, and/or penetrate a positively-charged tissue; a relatively positively charged nanoparticle is used to preferentially localize to, accumulate, and/or penetrate a negatively-charged tissue. For example, localization of the cargo-containing nanopiece is at least 10%, 20%, 50%, 75%, 2-fold, 5-fold, 8-fold, 10-fold or more to a target tissue compared to the level of localization/delivery of the cargo in the absence of the nanoparticle. Thus, the nanopieces are selectively localized to a desired bodily tissue and deliver the cargo there.
The drug or agent delivered comprises a diagnostic reagent or a therapeutic compound. In one example, a net positive charge comprises a Zeta potential in the range of +0 mV and +60 mV (e.g., 0.1 mV, 1, 5, 10, 20, 30, 45, 60 mV); exemplary negatively charged tissues include cartilage tissue or a chondrocyte cell. In another example, a charge comprising a Zeta potential in the range of −60 mV and +30 mV (e.g., −60, −50, −40, −30, −20, −10, 1, 10, 20, 30 mV) is used to selectively or preferentially target positively charged tissues; exemplary positively charged tissues include neuronal tissue or a neuron.
Also within the invention is a system for selective drug delivery to a bodily tissue comprising a nanoparticle composition comprising a cargo compound, the composition being sized to localize or penetrate a target tissue. The nanoparticle is at least 0.1 nm in at least one dimension. For example, a size of ≦150 nm (e.g., 0.1, 10, 25, 50, 75, 100, 125, 150 nm) in at least one dimension localizes to or penetrates synovium, ocular tissue, dermatologic tissue, mucosal tissue, or pulmonary tissue, a size of ≦100 nm (e.g., 0.1, 10, 25, 50, 75, 100 nm) in at least one dimension localizes to or penetrates kidney tissue, or a size of ≦30 nm (0.1, 2, 5, 10, 20, 25, 30 nm) in at least one dimension localizes to or penetrates heart tissue. A size of ≦90 nm (0.1, 2, 5, 10, 25, 50, 75, 80, 90 nm) in at least one dimension localizes to or penetrates cartilage with inflammation or defect, and a size of ≦30 nm (0.1, 2, 5, 10, 20, 25, 30 nm) in at least one dimension localizes to or penetrates healthy, intact cartilage.
The system or method includes the treatment of joint disorders those affecting articulating joints, e.g., injury-induced osteoarthritis as well as autoimmune diseases affecting joint tissue such as rheumatoid arthritis. The compositions and methods of the invention further provide a solution to long standing challenges in the treatment of diseases and/or disorders affecting the epithelial, connective, muscles and/or nervous tissues in the body. The invention provides methods of introducing a therapeutic or diagnostic agent into a cell or tissue or tissue matrix using rosette nanotubes or components of rosette nanotubes. Embodiments of the present disclosure include the formation of a composite or complex or combination of one or more agents, such as therapeutic or diagnostic agents, and a rosette nanotube or a component of a rosette nanotube, where the one or more agents are attached to or otherwise bound to the rosette nanotube or component of a rosette nanotube. Embodiments of the present disclosure are further directed to a product made by the process of mixing together rosette nanotubes as described herein or modules forming rosette nanotubes as described herein and one or more agents in aqueous media under conditions which cause the rosette nanotubes or components of rosette nanotubes to combine with the one or more agents to form a complex or combination in aqueous media where the one or more agents are attached or otherwise bound through steric, ionic, or other forces to the rosette nanotube a component of a rosette nanotube. According to one aspect, the one or more agents are bound by noncovalent forces.
The nanopiece compositions are made from nanotubes made from modules that self-assemble, e.g., compounds comprising Formula I (module I) or compounds comprising Formula II (module II). Nanotubes according to the present disclosure include compounds of Formula I below:




wherein X is CH or nitrogen; n is an integer of, 1, 2, 3, or 4; R2 is hydrogen or a linker group for example (CH2)n or other linker groups described herein; Y is absent when R2 is hydrogen or is an amino acid or polypeptide having an amino group covalently bound to an α-carbon of the amino acid and the amino group is covalently bound to the linker group R2; and R1 is hydrogen or an aliphatic moiety, such as alkyl, straight or branched chain, saturated or unsaturated; and salts thereof. Preferably R1 is C1 to C10 alkyl, C1 to C5 alkyl, C1 to C3 alkyl, or methyl. For example, one subset of compounds of formula (I) includes those in which X is nitrogen. In another example, one subset of compounds of formula (I) includes those in which (CH2)n is the linker group. In another embodiment, one subset of compounds of formula (I) includes those in which (CH2)n, is the linker group and n is 2. In another example, one subset of compounds of formula (I) includes those in which Y is an amino acid selected from lysine, arginine and histidine. In another embodiment, one subset of compounds of formula (I) includes those in which X is nitrogen, (CH2)n is the linker group, n is 2 and Y is an amino acid selected from lysine, arginine and histidine.
Compounds within the scope of the invention include those where the Y group can be connected to the linker group either by the amino group or the carboxyl group of the amino acid or polypeptide. An exemplary linker group is shown in the formula below.




An exemplary module within the scope of formula I is shown in FIG. 1 along with a schematic representation of a nanotube and an image of nanotubes formed from the exemplary module.
Alternative linker groups R2 can join the Y group to the carbon of the (CH2)n group or the N atom either by the amino group or the carboxyl group of the amino acid or polypeptide.
Alternative R2 groups within the scope of the present disclosure are selected from a group comprising:







wherein Y is absent.

Compounds of Formula I can be prepared by the methods described in U.S. Pat. No. 6,696,565 hereby incorporated by reference herein in its entirety alone or combined with methods known to those of skill in the art. Additional description is provided in U.S. Pat. No. 8,795,691 and/or U.S. Patent Publication 20140171482 (U.S. Ser. No. 13/977,138), each of which is hereby incorporated by reference. Rosette nanotubes are made by assembly of compounds of Formula (I).

Exemplary compounds of Formula I are shown below:




Modules according to the present disclosure also include compounds of Formula II below:




wherein X is CH or nitrogen; R2 is hydrogen or a linker group for example (CH2)n where n is an integer of, 1, 2, 3, or 4 or (CH2)3CO other linker groups described herein; Y is absent when R2 is hydrogen or is an amino acid or polypeptide having an amino group covalently bound to an α-carbon of the amino acid and the amino group is covalently bound to the linker group R2; and R1 is hydrogen or an aliphatic moiety, such alkyl, straight or branched chain, saturated or unsaturated; and salts thereof. For example, one subset of compounds of formula (II) includes those in which X is nitrogen. In another example, one subset of compounds of formula (II) includes those in which (CH2)n is the linker group. In another embodiment, one subset of compounds of formula (II) includes those in which (CH2)n is the linker group and n is 2. In another example, one subset of compounds of formula (II) includes those in which Y is an amino acid selected from lysine, arginine and histidine. In another embodiment, one subset of compounds of formula (II) includes those in which X is nitrogen, (CH2)n is the linker group, n is 2 and Y is an amino acid selected from lysine, arginine and histidine.
Preferably R1 is C1 to C10 alkyl, C1 to C5 alkyl, C1 to C3 alkyl, or methyl. An exemplary linker group is shown in the formula below.




Compounds within the scope of the present disclosure include those where the Y group can be connected to the linker group either by the amino group or the carboxyl group of the amino acid or polypeptide. Alternative R2 groups within the scope of the present disclosure are selected from a group comprising:








wherein Y is absent. TBL structures are made by the assembly of compounds of Formula (II).

Exemplary compounds of Formula II are shown below:










In some embodiments, compounds of formula II comprise amino acid functional group constructs. These compounds contain functional groups present in natural occurring amino acid side chains or may contain the entire amino acid side chain. For example, the lysine functional group constructs contains the entire amino acid side chain functionality (—CH2CH2CH2CH2NH3+), whereas the histidine functional group constructs contains the entire side chain or only contains the heteroaryl imidazole group present in histidine.
In some embodiments, compounds of formula II comprise amino acid constructs. These compounds contain the entire the amino acid or may contain modified and/or unnatural amino acids. For example, the lysine amino acid analog contains the entire amino acid functionality of lysine, whereas the histidine amino acid constructs contains a modified histidine amino acid.
In some embodiments the compounds of formula II are preferred over the compounds of formula I.
In some embodiments the compound of formula II is the Lysine Functional Group Construct




In some embodiments, the nanoparticles are constructed from lipid and/or polymeric components.
A three-dimensional representation of such modules is shown in FIG. 65. Embodiments further include delivering the composite into living cells. Embodiments further include a method of treating an individual requiring treatment comprising administering a complex of a rosette nanotube or a component of a rosette nanotube and one or more therapeutic agents to the individual in a manner to introduce the complex into cells or tissues of the individual. Embodiments further include a method of diagnosing an individual requiring diagnosis comprising administering a complex of a rosette nanotube or a component of a rosette nanotube and one or more diagnostic agents to the individual in a manner to introduce the complex into cells or tissues of the individual.
Rosette nanotubes or RNTs include nanotubes formed from modules having twin bases with a linker or TBL. Such rosette nanotubes may be referred to herein as “TBLs.” According to this aspect, the agent is delivered into the cell. According to one aspect, the agent is released from the rosette nanotube after entry into the cell. According to an additional aspect, the agent remains attached to, bound to, or complexed with or combined with the rosette nanotube or component of a rosette nanotube.
Lipid nanoparticles comprise a lipid core and surfactant, in which the lipid core may include fatty acids, acrylglycerols, steroids, waxes, and mixtures of all above; and surfactants may contain a positively charged amino group, negatively charged phosphate or carboxylic acid. According to one aspect, a complex is produced by combining modules of a self-assembled rosette nanotube and one or more agents in media where the modules self-assemble into a rosette nanotube or components of a rosette nanotube which incorporates the one or more agents to form a complex of a rosette nanotube or component of a rosette nanotube and the one or more agents. According to an additional aspect, a complex is produced by combining a self-assembled rosette nanotube and one or more agents in media whereupon the one or more agents are incorporated into the rosette nanotube to form a complex of a rosette nanotube and one or more agents. The complex may then be contacted to cells whereupon the complex enters the cells. Without wishing to be bound by scientific theory, it is believes that the complex may enter cells by endocytosis. According to certain embodiments, the cells may be transformed cells, recombinant cells, malignant cells, or cells from primary cell lines. The transfection method may be performed on cells in vitro or in vivo.
The modules may be any of those known to persons of ordinary skill in the art such as G^C motifs and A^T motifs, unmodified or modified to include moieties or side chains, which self-assemble into helical rosette nanotubes. According to one embodiment, modules are placed into an aqueous medium where they self assemble into a substructure such as a ring structure, such as a rosette, and the ring structures then self-assemble by stacking one on top of another to form a tubular structure, commonly referred to as a nanotube. Such modules, substructures and nanometer scale molecular structures and their self-assembly is described in U.S. Pat. No. 6,696,565, Fenniri et al, J. Am. Chem. Soc. 2001, 123, 3854-3855, Moralez et al., J. Am. Chem. Soc., 2005, 127, 8307-8309, Fine et al., International Journal of Nanomedicine 2009:4 91-97; and Zhang et al., Biomaterials 2009; 30(7):1309-1320 each of which are hereby incorporated by reference in their entireties for all purposes.
Rosette nanotubes of the present disclosure are very stable in water and lack virus-related safety concerns and toxicity at amounts of about 1 μg/ml. See Int. J. Nanomedicine, 2008, 3(3):373-383; Small. 2008, 4(6):817-823; and Am. J. Physiol Lung Cell Mol. Physiol. 2005, November, 289(5):L698-708 each of which are hereby incorporated by reference in their entireties.
According to one aspect of the present disclosure, methods are provided where the self-assembly of precursors or modules incorporates the agent into or otherwise complexes the agent with, the self-assembled rosette nanotube or components of the rosette nanotube. According to another aspect, fully assembled rosette nanotubes can be incubated with one or more or a plurality of agents and the one or more or plurality of agents can complex with the fully assembled rosette nanotube to form a composite. According to one further aspect, the one or more or plurality of agents are joined to or bound to the self-assembled rosette nanotube through steric, ionic, van der Waals, dispersion or other noncovalent interactions to form a rosette nanotube or component of a rosette nanotube and agent complex useful as a complex to be administered to an individual. In another aspect of the invention, the agents comprise a therapeutic agent such as nucleic acid, peptide or small molecule. In a further aspect of the invention, the therapeutic agent comprises an IL-1 receptor antagonist. In yet a further aspect of the invention, the agent comprises a diagnostic agent such as a molecular probe or a molecular beacon. For example, the molecular beacon or probe comprises MMP-13 or ADAMTS-5.
According to certain aspects of the invention, a method for treating joint disease comprises administration of an effective amount of a composition containing a nanotube rosette-agent complex. Such a method of diagnosing joint disease comprises administration of an effective amount of a composition containing a nanotube rosette-agent complex. Another aspect of the invention comprises joint disease such as autoimmune, degenerative, inflammatory, infectious, cancerous, viral, fungal, injured, trauma, genetic, trauma, mechanical, nutritional or mal-alignment derived. Another embodiment of the invention describes joint disease comprising rheumatoid arthritis, osteoarthritis, juvenile onset of rheumatoid arthritis (JRA), psoriatic arthritis, reactive arthritis, septic arthritis, tendinitis, or herniation. Therapeutic agents are used to treat joint disease, e.g., such agents include analgesic agents, anti-inflammatory agents, immunosuppresive agents, antifungal agents, antibiotic agents, lurbicants, anti-cancer agents, NMDA receptor antagonists, or antiviral agents.
According to certain aspects of the invention, a method for treating tissue and/or organ disease comprises administration of an effective amount of a composition containing a nanotube rosette-agent complex. Such a method of diagnosing tissue and/or organ disease comprises administration of an effective amount of a composition containing a nanotube rosette-agent complex. Another aspect of the invention comprises a tissue and/or organ disease such as autoimmune, degenerative, inflammatory, infectious, cancerous, viral, fungal, injured, trauma, genetic, trauma, mechanical, nutritional or mal-alignment derived. Another embodiment of the invention describes tissue and/or muscle disease comprising the eye, skin, brain, spine, intestine, kidney, liver, and stomach. Another aspect of the invention describes therapeutic agents to treat joint, tissue and/or organ disease, e.g., agents include analgesic agents, anti-inflammatory agents, immunosuppresive agents, antifungal agents, antibiotic agents, lurbicants, anti-cancer agents, NMDA receptor antagonists, or antiviral agents.
According to certain aspects, rosette nanotubes are functionalized with a nucleic acid, such as DNA or small RNA to form a complex, for example RNA is bound to the rosette nanotube, the complex is translocated into a cell or tissue, and the intracellular small RNA (e.g., siRNA) is present within the cell in an amount sufficient for gene silencing resulting in the inhibition of the production of target proteins. In this aspect, the rosette nanotube is a delivery vehicle or carrier for the small RNA into a cell for RNA interference purposes. Alternatively, the nucleic acid can be expressed by the cell. For example, the cell comprises synoviocytes or chondrocytes. Alternatively, the target tissue is cartilage. According to certain aspects, methods and technologies are provided to process and assemble rosette nanotubes (RNTs) for cargo delivery for diagnostic and therapeutic purpose. Methods are directed to achieve inter-/intra-cellular delivery in vitro and in vivo. According to certain aspects, a complex of rosette nanotubes (RNTs) and cargo agents are prepared. The cargo agents include diagnostic molecules, for instance, oligomer based molecular beacons; or therapeutic molecules such as nucleic acid, peptide, or small molecules. Such diagnostic agents and therapeutic agents are well known to those of skill in the art. Such incorporation between RNTs and the cargo reagent are facilitated by electrostatic force, π-π interactions or hydrophilic/hydrophobic effects to form relatively stable entities, which are referred to herein as “Nanopieces”. According to certain aspects, methods are provided to make rosette nanotubes of certain size (with or without an agent (e.g., cargo composition) that are suitable for trans-matrix e.g., extracellular matrix, tissue delivery. For example, methods are provided for altering at least one dimension or other parameter of Nanopieces such as width to infiltrate the pore size of the target tissue matrix.
According to certain aspects, methods and technologies are provided to process and assemble rosette nanotubes (RNTs) for cargo delivery for diagnostic and therapeutic purpose. Methods are directed to achieve inter-/intra-cellular delivery in vitro and in vivo. According to certain aspects, a complex of rosette nanotubes (RNTs) and cargo agents are prepared. The cargo agents include diagnostic molecules, for instance, oligomer based molecular beacons; or therapeutic molecules such as nucleic acid, peptide, or small molecules. Such diagnostic agents and therapeutic agents are well known to those of skill in the art. Such incorporation between RNTs and the cargo reagent are facilitated by electrostatic force, π-π interactions or hydrophilic/hydrophobic effects to form relatively stable entities, which are referred to herein as “Nanopieces”. According to certain aspects, methods are provided to make rosette nanotubes of certain size with or without an agent that are suitable for trans-matrix tissue delivery. For example, methods are provided for altering at least one dimension parameter of Nanopieces such as width to infiltrate the pore size of the target tissue matrix.
According to certain aspects, methods are provided for making rosette nanotubes of certain lengths and size parameters such as 1) before assembly, controlling the length and bundle of RNTs via changing physical and/or chemical conditions such as temperature, molecule motion/vibration (like sonication) and pH; 2) during assembly, adjusting assembly conditions via changing physical and/or chemical conditions including concentrations, pH and ionic strength to enhance/reduce the formation and stacking of Nanopieces; 3) after assembly, breaking long or stacked Nanopieces by via changing physical and/or chemical conditions including enhancing molecule motion/vibration (like sonication).
According to certain aspects, methods are provided for trans-matrix/tissue delivery or a complex of a rosette nanotube or component or piece thereof by controlling the ratio between RNTs and cargo reagents so that the forming Nanopieces present surface charges that are suitable for attraction, localization, penetration, or retention in the tissue or one or more cells of the tissue. For example, since many tissues or cells contain negatively charged molecules (like proteoglycan), positively charged RNT can be fabricated and used to assemble with negatively charged nucleic acid cargo in certain ratios, resulting in a positive charged Nanopiece for delivery. In this manner, Nanopieces localize to, bind to, and accumulate onto/into the matrix/tissue resulting in much longer retention time to achieve more effective delivery. Therefore, the highly effective and versatile trans-matrix/tissue delivery was achieved by processed Nanopieces. The term “Nanopiece” may be used herein to refer to rosette nanotubes which may be processed into certain dimensions or components of rosette nanotubes.
According to certain aspects, methods are provided for the use of rosette nanotubes or Nanopieces for diagnostic applications insofar as molecular probes can be delivered via Nanopieces to detect a specific gene expression (or protein activity). By co-delivery of a negative control for non-specific signal and an internal positive control, a target gene expression can be accurately diagnosed in a real-time, in-situ and non-invasive manner.
According to certain aspects, therapeutic applications are envisioned, such as knocking down one or multiple disease gene expression (such as via siRNA, miRNA or anti-sense delivery), e.g., inhibiting the expression of one or more genes or gene products associated with aberrantly high expression in a disease state compared to a normal state up-regulating one or multiple beneficial gene/protein (such as via DNA, mRNA or protein delivery); or through a combination of both.
According to certain aspects, methods are provided for making rosette nanotubes of certain lengths and size parameters such as 1) before assembly, controlling the length and bundle of RNTs via changing physical and/or chemical conditions such as temperature, molecule motion/vibration (like sonication) and pH; 2) during assembly, adjusting assembly conditions via changing physical and/or chemical conditions including concentrations, pH and ionic strength to enhance/reduce the formation and stacking of Nanopieces; 3) after assembly, breaking long or stacked Nanopieces by via changing physical and/or chemical conditions including enhancing molecule motion/vibration (like sonication).
According to certain aspects, method are provided for trans-matrix/tissue delivery or a complex of a rosette nanotube or component or piece thereof by controlling the ratio between RNTs and cargo reagents so that the forming Nanopieces present surface charges that are suitable for retention in the tissue. For example, since many tissues or cells contain negatively charged molecules (like proteoglycan), positively charged RNT can be used to assemble with negatively charged nucleic acid cargo in certain ratios, resulting in a positive charged Nanopiece for delivery (see Table 1). In this manner, Nanopieces associate with, bind to and/or accumulate onto/into the matrix/tissue resulting in much longer retention time to achieve more effective delivery. Therefore, the highly effective and versatile trans-matrix/tissue delivery was achieved by processed Nanopieces.
According to certain aspects, methods are provided for the use of rosette nanotubes or Nanopieces for diagnostic applications insofar as molecular probes can be delivered via Nanopieces to detect a specific gene expression (or protein activity). By co-delivery of a negative control for non-specific signal and an internal positive control, a target gene expression can be accurately diagnosed in a real-time, in-situ and non-invasive manner.
According to certain aspects, therapeutic applications are envisioned, such as knocking down one or multiple disease gene expression (such as via siRNA delivery); up-regulating one or multiple beneficial gene/protein (such as via DNA, mRNA or protein delivery); or through a combination of both.
According to certain aspects, depending on the processing conditions, different sizes of rosette nanotubes, e. g. Nanopieces can be created for different delivery proposes, such as to enter a cellular or tissue matrix. For example, cartilage tissue matrix has about 60 nm mesh size of the collagen II fibrillar network (Comper et al in Cartilage: Molecular Aspects (eds Hall, B. & Newman, S.) 59-96 (CRC Press, Boston, 1991)) and about 20 nm spacing between the side chains of the proteoglycan network (Torzilli et al J. Biomech. 30, 895-902 (1997)). Nanopieces with small sizes (at least one dimension smaller than 60 nm and/or 20 nm) showed excellent efficiency and function in intra-cartilage matrix delivery of siRNA. Secondly, through adjusting the ratio between RNTs and cargo reagents, overall positive charged surface enabled Nanopieces to adhere with negatively charged matrix/tissue components resulting longer retention time. Thirdly, Nanopieces can deliver a variety of cargo types and can deliver multiple cargo reagents at the same time. Fourthly, using non-covalent or covalent coating on Nanopieces can achieve a longer stability in the systemic circulation and penetrate into the targeted tissue matrix and/or organ more efficiently. Lastly, processed Nanopieces demonstrated successful delivery under conditions: in vitro, ex vivo and in vivo. Therefore, methods are provided for the use of Nanopieces for trans-matrix/tissue delivery.
According to certain aspects, complexes of rosette nanotubes or components of nanotubes or Nanopieces and agents can be used for research purposes as well as used for an effective delivery agent (especially in vivo) for molecular diagnosis and therapeutics. According to certain aspects, complexes of rosette nanotubes or components of nanotubes or Nanopieces and agents can be used for therapeutic purposes for treating various diseases, such as by delivery of interleukin-1 receptor antagonist (IL-1Ra), the natural protein inhibitor of IL-1, to modulate IL-1-based inflammation as a therapy for arthritis. For example, the cargo comprises IL-1R SiRNA. Complexes of rosette nanotubes or components of nanotubes or Nanopieces and agents can be used to deliver siRNA to knockdown the disease protein to achieve effective treatment.
According to certain aspects, complexes of rosette nanotubes or components of nanotubes or Nanopieces and agents can be used for diagnostics, such as by delivery of molecular probes or molecular beacons. Methods are provided to deliver molecular beacons into chondrocytes inside cartilage matrix as well as tissues and/or organs such as heart, stomach, kidney, liver, lung, spleen, brain, intestine, spine, rib cage, and limb. With co-delivery of multiple molecular beacons to detect disease gene expression as target, non-specific signal as negative control and house-keeping gene as internal positive control, target gene expression level can be quantified in a real-time, in-situ and non-invasive manner.
Embodiments of the present disclosure are directed to complexes of a self-assembled rosette nanotube and one or more or a plurality of agents. Such agents include biologically active agents and/or diagnostic agents. The complexes are administered to an individual where the biologically active agent and/or diagnostic agent are delivered to a site within the individual, including into the cell of an individual, and are made available for therapeutic or diagnostic purposes. According to one aspect, the agent dissociates from the rosette nanotube to treat an individual or to provide a diagnostic capability. According to an additional aspect, the agent remains attached to, bound to, or complexed with or combined with the rosette nanotube.
According to one aspect, a delivery complex is produced by combining modules of a self-assembled rosette nanotube and one or more agents, such as therapeutic or diagnostic agents, in media where the modules self-assemble into a rosette nanotube which incorporates the one or more agents to form a complex of a rosette nanotube and the one or more agents. According to an additional aspect, a delivery complex is produced by combining a self-assembled rosette nanotube and one or more agents, such as therapeutic or diagnostic agents, in media whereupon the one or more agents are incorporated into the rosette nanotube to form a complex of a rosette nanotube and one or more agents. The delivery complex may then be administered to an individual for therapeutic or diagnostic purposes. It is a further object of the present invention to create complexes of agents rosette nanotubes or components of rosette nanotubes that can be delivered into target cells and intracellular matrices where the agent can function. It is a further object of the present invention to provide methods of treating individuals using a delivery system of a complex of rosette nanotubes or components of rosette nanotubes and agents, where the agent enters the cell. These and other objects, features, and advantages of the invention or certain embodiments of the invention will be apparent to those skilled in the art from the following disclosure and description of exemplary embodiments.
It is a further object of the present invention to create complexes of agents rosette nanotubes or components of rosette nanotubes that can be delivered into target cells and intracellular matrices where the agent can function. It is a further object of the present invention to provide methods of treating individuals using a delivery system of a complex of rosette nanotubes or components of rosette nanotubes and agents, where the agent enters the cell. Thus, the invention encompasses a composition comprising a cargo molecule and a nanostructure comprising Formula I or Formula II for selective, e.g., preferential, delivery of a therapeutic drug or diagnostic agent to a target bodily tissue. Alternatively, the non-structure comprises a lipid or a polymer rather than a compound or Formula I or II.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an illustration showing an assembly between RNTs with siRNA.
FIG. 2 is an illustration showing an assembly between RNTs with plasmid DNA.
FIG. 3 is an illustration showing an assembly between RNTs with Matrilin-3.
FIG. 4 illustrates scheme 1, which displays an assembly mechanism and processing approaches.
FIG. 5A is a bar graph of the size distribution of Nanopieces assembled under standard conditions.
FIG. 5B is a bar graph of the width distribution of Nanopieces assembled under standard conditions.
FIG. 6A is a bar a graph of the size distribution of Nanopieces processed before assembly (quench).
FIG. 6B is a bar graph of the width distribution of Nanopieces processed before assembly (quench).
FIG. 7A is a bar graph of the size distribution of Nanopieces processed before assembly (sonication).
FIG. 7B is a bar graph of the width distribution of Nanopieces processed before assembly (sonication).
FIG. 8A is a bar graph of the size distribution of Nanopieces processed during assembly (increasing ionic strength).
FIG. 8B is a bar graph of the width distribution of Nanopieces processed during assembly (increasing ionic strength).
FIG. 9A is a bar graph of the size distribution of Nanopieces processed after assembly (increasing sonication time).
FIG. 9B is a bar graph of the width distribution of Nanopieces processed after assembly (increasing sonication time).
FIG. 10 is a series of images showing Nanopieces assembled before processing (Left) and after processing with sonication (Right) were delivered into cells.
FIG. 11 is a graph showing the Zeta potential (reflecting surface charge) of Nanopieces with different RNT/siRNA ratios.
FIG. 12 shows a series of images and a bar graph illustrating cartilage binding with RNTs, fluorescence labeled siRNA and RNT/siRNA Nanopieces on articular cartilage.
FIG. 13 is a series of images showing fluorescence labeled siRNA/RNT Nanopieces were delivered into porcine cartilage (Right) compared with controls (siRNA only).
FIG. 14 is a series of images showing effective delivery of processed GAPDH molecular beacon/RNT Nanopieces into mouse cartilage tissue matrix and inside chondrocytes.
FIG. 15 is a series of images showing effective delivery of processed GAPDH molecular beacon/RNT Nanopieces into human cartilage tissue matrix and inside chondrocytes.
FIG. 16 is a series of images showing effective delivery of processed GAPDH molecular beacon/RNT Nanopieces into chicken cartilage tissue matrix and inside chondrocytes.
FIG. 17 is a graph showing functional delivery of processed MATN3 siRNA/RNT Nanopieces into mouse cartilage tissue matrix and inside chondrocytes.
FIG. 18 is a graph showing functional delivery of processed MATN3 siRNA/RNT Nanopieces into mouse cartilage tissue matrix and inside chondrocytes.
FIG. 19 is a graph showing functional delivery of processed miRNA365/RNT Nanopieces into human cartilage tissue matrix and inside chondrocytes.
FIG. 20 is a graph showing functional delivery of processed miRNA365/RNT Nanopieces with and/or without PEG into human cartilage tissue matrix and inside chondrocytes in the serum and serum-free medium.
FIG. 21 is an image showing injection of reagents into mouse knee joints.
FIG. 22 is a series of images showing fluorescent signals in mouse cartilage tissue matrix over time by injecting processed RNT/beacon Nanopieces.
FIG. 23 is a series of images showing fluorescent signals in mouse cartilage tissue matrix over time by injecting molecular beacon only.
FIG. 24 is a graph showing quantitative fluorescent signals in mouse cartilage tissue matrix over time.
FIG. 25 is a graph and an image showing in vivo delivery of processed RNT/beacon Nanopieces into rat cartilage tissue matrix and inside chondrocytes compared with beacon only.
FIG. 26 is a series of images and a bar graph showing qualitative (Left) and quantitative (Right) in vivo delivery of processed RNT/beacon Nanopieces into rat cartilage tissue matrix and inside chondrocytes compared with beacon only.
FIG. 27 is an image showing injection of reagents into baby mouse joints.
FIG. 28 is a series of images showing histology sections of cartilage delivered with RNTs only (Top), beacon only (Middle) and RNT/beacon Nanopieces (Bottom).
FIG. 29 is a series of images showing in vitro validation of MMP-13 molecular beacon.
FIG. 30 is an image showing comparison of fluorescence signal between DMM and Sham knees (dark grey is GAPDH; light grey is MMP-13).
FIG. 31 is a graph showing DMM/Sham MMP-13 signal over time.
FIG. 32 is a graph showing DMM knee relative MMP-13 expression level.
FIG. 33 is a series of graphs showing relative IL-1R, MMP-13, MMP-9 and Col II gene expression level after therapeutically knock down of IL-1R.
FIG. 34 is a series of images showing histology (medium grey staining is proteoglycan) and immunohistochemistry (dark grey staining is epitope from aggrecan cleavage) of mouse knee joints. ADAMTS-5 siRNA/Nanopiece greatly inhibited cartilage degeneration and Aggrecan cleavage with cytokine stimulation.
FIG. 35 is a series of images showing histology of mouse knee joints. ADAMTS-5 siRNA/Nanopiece greatly inhibited cartilage degeneration after DMM surgery.
FIG. 36 is a graph showing histology evaluation of mouse knee joints. ADAMTS-5 siRNA/Nanopiece prevents osteoarthritis progression after DMM surgery.
FIG. 37 is a series of images showing a comparison with fluorescence signal from scrambled molecular beacon, signal from MMP-13 molecular beacon indicating the area of MMP-13 expression and articular cartilage degeneration.
FIG. 38 is an image of histology staining of a mouse knee joint after DMM surgery. The area of cartilage degeneration is the same as what was indicated by MMP-13 molecular beacon.
FIG. 39 is a series of images showing GAPDH and Scrambled molecular beacon delivered by Nanopieces into chondrocytes with stimulation.
FIG. 40 is a series of images showing GAPDH and ADAMTS-5 molecular beacon delivered by Nanopieces into chondrocytes without stimulation.
FIG. 41 is a series of images showing GAPDH and ADAMTS-5 molecular beacon was delivered by Nanopieces into chondrocytes with stimulation.
FIG. 42 is an image of fluorescence signal of ADAMTS-5 molecular beacon in DMM and Sham knees on day 6 after surgery.
FIG. 43 is a graph showing fluorescence signal ratio of ADAMTS-5 molecular beacon in DMM knees over Sham knees after surgery.
FIG. 44 is a series of images illustrating immunohistochemistry results (staining is epitope from aggrecan cleavage) of human articular cartilage. ADAMTS-4 siRNA and combination of ADAMTS-4&5 siRNA/Nanopieces greatly inhibited Aggrecan cleavage with cytokine stimulation.
FIG. 45 is a series of images showing histology results (staining is proteoglycan) of human articular cartilage. ADAMTS-4 siRNA and combination of ADAMTS-4&5 siRNA/Nanopieces greatly inhibited cartilage degradation with cytokine stimulation.
FIG. 46 is a series of images showing immunohistochemistry results (staining is epitope from aggrecan cleavage) of mouse knee joints. ADAMTS-5 siRNA/Nanopieces greatly inhibited Aggrecan cleavage after DMM surgery.
FIG. 47 is a graph showing cell toxicity studies of RNTs purified using HPLC chromatography with HCl or TFA as a modifier.
FIG. 48 is a series of images showing the conversion of nanotubes to nanorods.
FIG. 49 is a series of images showing the generation of Nanopieces before and after “processing-2”.
FIG. 50 is a graph showing quantitative analysis of fluorescence signal in mouse knee.
FIG. 51 is a scheme showing molecular beacon (MB) technology.
FIG. 52 is a scheme showing trans matrix delivery of Nanopieces into chondrocytes.
FIG. 53 is flow design of self-assembly, processing-1, processing-2 to yield nanopieces.
FIG. 54 is a graph showing MMP expression increase 4 days after surgery.
FIG. 55 is a graph showing MMP-expression increase 11 days after surgery.
FIG. 56 is a series of graphs an images showing Nanopieces size and morphology with increasing sonication power.
FIG. 57 is a scatter plot of Nanopieces size and morphology with increasing sonication power.
FIG. 58 is a line graph showing the stability of Nanopieces with different molar-excess ratios of PEG.
FIG. 59 is a line graph showing the stability of Nanopieces with and without non-covalent linked PEG.
FIG. 60 is an image showing the delivery of small Nanopieces into articular cartilage to result in fluorescence comparted to controls (MB only).
FIG. 61 is an image showing the delivery of both large and small Nanopieces into synovium to result in fluorescence compared with controls (MB only).
FIG. 62 is an image showing the decreased liver capture with small Nanopieces compared with lipid vehicles.
FIG. 63 is a bar graph showing the decreased liver capture with small Nanopieces compared to lipied vehicles.
FIG. 64 is a bar graph showing increased delivery into tissues or organs with dense matrix with small Nanopieces.
FIG. 65 is an illustration showing a structure of RNT. It is a long tubular structure with outside diameter of 3.5 nm, and inside diameter of 1.1 nm.
FIG. 66 is a series of images showing that cells with Nanopiece (RNT or TBL) delivery maintain normal cell morphology, indicating excellent biocompatibility of Nanopiece; while delivery with lipid-based vehicles led to abnormal cell morphology and large amount of debris, suggesting cyto-toxicity of lipid-based vehicles.
FIG. 67 is a bar graph showing PCR results of IL-1R expression levels of large and small lipid nanoparticles (* p<0.05 compared to negative controls and large lipid nanoparticle).
FIG. 68 is a bar graph showing PCR results of IL-1R expression levels of large and small polymer nanoparticles (* p<0.05 compared to negative controls and large polymer nanoparticle).
FIG. 69 shows amino acids containing hydrophilic side chains, hydrophobic side chains, and electrically charged side chains, respectively.



DETAILED DESCRIPTION
The compositions and methods of the invention provide compositions and methods for preferential targeting of tissues to delivery therapeutic agents. The structures, e.g., nanopieces, are constructed to comprise a charge and/or size such that the structures preferentially associate with or bind to specific bodily tissues. For example, the invention provides methods for the delivery of Nanopieces and their cargo to/into joints, tissue and/or organs. A successful delivery into cells does not always necessarily mean that a successful delivery into tissue is achieved to obtain an efficacious therapeutic or diagnostic outcome. One major reason is that tissues unlike cells have an extracellular matrix. For example, Nanopieces with large size or inappropriate surface charge may not penetrate the tissue efficiently enough to cause a therapeutic or diagnostic response. Drug molecules released from nanotubes prior to tissue penetration do not diffuse into enough depth of the tissue to reach a significant amount of cells. The invention solves such problems and provides methods to package drug molecules within nanotubes/nanorods that are selectively designed to alter their surface charge and/or their size to be small enough to penetrate the tissue matrix. So in this manner it is not the drug molecules that are released from the nanotubes and then diffuse into the tissue but it is the actual Nanopieces/nanorods (containing cargo, e.g., drug) that penetrate the tissue. The invention further provides methods of processing nanotubes/nanorods to control of size and other properties of Nanopieces (like surface charge and coating), in order to efficiently deliver their cargo into joints, tissues and/or organs to achieve an effective therapy or diagnosis. These Nanopieces (Nanopieces) may contain nucleic acid, peptides, proteins and aromatic or negatively charged small molecules. Because different tissues have different surface charge, it is important to control the surface charge of Nanopieces via the ratio of delivery cargos and amount of nanorods. Nanopieces, which are too large may have difficulties in penetrating the tissue matrix and improper surface charge of Nanopieces may be repulsive to the target tissue matrix or perhaps the Nanopieces are not stable in the bodily fluids or blood. The table below describes exemplary nanopieces for preferential localization to and delivery to exemplary bodily tissues.
Selective Delivery of Nanopieces to Target Tissues












TABLE 1








Processing






Nanopiece
details to


Target
Nanopiece
Charge
achieve desired
Preferred


Tissue/Cell
Structure
(Zeta
length/width/
payload/
Other/


Type
Size
potential)
charge*
cargo
notes







Cartilage/
General range: at
General
Ratio: 4.4~30 μg
siRNA,
Negatively


chondrocyte
least one dimension
range:
RNTs per
other
charged



between 1 nm and 90 nm
between +0 mV
0.1 nmol RNA
nucleic



Preferred range: at
and
(Preferred ratio:
acids,



least one dimension
+60 mV
6.6~20 μg RNTs
molecular



between 1 nm and 30 nm
Preferred
per 0.1 nmol
beacons and




range:
RNA)
peptides/




between +8 mV
Sonication
proteins




and
power:
(ADAMTS-




+40 mV
10%~100% (for
5 siRNA,





a 700 W
MMP-13





sonicator)
oligo





Sonication time:
molecular





10 s~30 mins
beacon,





Ionic strength of
IL-1Ra





assembly
protein)





solution:





0~308 mmol/L





At least one of





pre-processing





methods (such





as heating,





sonication or





quench):





required


Synovium
General range: at
General
Ratio: 4.4~30 μg
siRNA,



least one dimension
range:
RNTs per
other



between 1 nm and
between +0 mV
0.1 nmol RNA
nucleic



150 nm
and
(Preferred ratio:
acids,



Preferred range: at
+60 mV
4.4~20 μg RNTs
molecular



least one dimension
Preferred
per 0.1 nmol
beacons and



between 10 nm and
range:
RNA)
peptides/



100 nm
between +0 mV
Sonication
proteins




and
power: 1~100%
(IL-1 or




+40 mV
(for a 700 W
TNF-α





sonicator)
siRNA,





Sonication time:
IL-1 or





5 s~30 mins
TNF-α oligo





Ionic strength of
molecular





assembly
beacon,





solution: no
IL-1Ra





requirement
protein)





At least one of





pre-processing





methods (such





as heating,





sonication or





quench): not





required


Neurons
General range: at
General
Ratio: 0.1~15 μg
siRNA,
Neurons



least one dimension
range:
RNTs per
other
generally



between 1 nm and
between −60 mV
0.1 nmol RNA
nucleic
positively



150 nm
and
(Preferred ratio:
acids,
charged



Preferred range: at
+30 mV
1~15 μg RNTs
molecular



least one dimension
Preferred
per 0.1 nmol
beacons and



between 10 nm and
range:
RNA)
peptides/



100 nm
between −40 mV
Sonication
proteins




and
power: 1~100%




+30 mV
(for a 700 W





sonicator)





Sonication time:





5 s~30 mins





Ionic strength of





assembly





solution: no





requirement





At least one of





pre-processing





methods (such





as heating,





sonication or





quench): not





required


Brain/BBB
General range: at
General
Ratio: 1~20 μg
siRNA,



least one dimension
range:
RNTs per
other



between 1 nm and
between −30 mV
0.1 nmol RNA
nucleic



100 nm
and
(Preferred ratio:
acids,



Preferred range: at
+40 mV
4.4~20 μg RNTs
molecular



least one dimension
Preferred
per 0.1 nmol
beacons and



between 1 nm and 30 nm
range:
RNA)
peptides/




between +8 mV
Sonication
proteins




and
power:




+40 mV
10~100% (for a





700 W sonicator)





Sonication time:





10 s~30 mins





Ionic strength of





assembly





solution:





0~308 mmol/L





At least one of





pre-processing





methods (such





as heating,





sonication or





quench):





required


Ocular
General range: at
General
Ratio: 4.4~30 μg
siRNA,


tissue
least one dimension
range:
RNTs per
other



between 1 nm and
between +0 mV
0.1 nmol RNA
nucleic



150 nm
and
(Preferred ratio:
acids,



Preferred range: at
+60 mV
4.4~20 μg RNTs
molecular



least one dimension
Preferred
per 0.1 nmol
beacons and



between 10 nm and
range:
RNA)
peptides/



100 nm
between +0 mV
Sonication
proteins




and
power: 1~100%




+40 mV
(for a 700 W





sonicator)





Sonication time:





5 s~30 mins





Ionic strength of





assembly





solution: no





requirement





At least one of





pre-processing





methods (such





as heating,





sonication or





quench): not





required


Derm tissue,
General range: at
General
Ratio: 4.4~30 μg
siRNA,


skin, etc.
least one dimension
range:
RNTs per
other



between 1 nm and
between +0 mV
0.1 nmol RNA
nucleic



150 nm
and
(Preferred ratio:
acids,



Preferred range: at
+60 mV
4.4~20 μg RNTs
molecular



least one dimension
Preferred
per 0.1 nmol
beacons and



between 10 nm and
range:
RNA)
peptides/



100 nm
between +0 mV
Sonication
proteins




and
power: 1~100%




+40 mV
(for a 700 W





sonicator)





Sonication time:





5 s~30 mins





Ionic strength of





assembly





solution: no





requirement





At least one of





pre-processing





methods (such





as heating,





sonication or





quench): not





required


Tumor
General range: at
General
Ratio: 0.1~30 μg
siRNA,
Tumors



least one dimension
range:
RNTs per
other
may be



between 1 nm and
between −60 mV
0.1 nmol RNA
nucleic
acidic



1200 nm
and
(Preferred ratio:
acids,



Preferred range: at
+60 mV
1~30 μg RNTs
molecular



least one dimension
Preferred
per 0.1 nmol
beacons and



between 10 nm and
range:
RNA)
peptides/



200 nm
between −30 mV
Sonication
proteins




and
power: 1~100%




+60 mV
(for a 700 W





sonicator)





Sonication time:





5 s~30 mins





Ionic strength of





assembly





solution: no





requirement





At least one of





pre-processing





methods (such





as heating,





sonication or





quench): not





required


Kidney
General range: at
General
Ratio: 4.4~30 μg
siRNA,



least one dimension
range:
RNTs per
other



between 1 nm and
between +0 mV
0.1 nmol RNA
nucleic



100 nm
and
(Preferred ratio:
acids,



Preferred range: at
+60 mV
4.4~20 μg RNTs
molecular



least one dimension
Preferred
per 0.1 nmol
beacons and



between 10 nm and
range:
RNA)
peptides/



200 nm
between +0 mV
Sonication
proteins




and
power: 5~100%




+40 mV
(for a 700 W





sonicator)





Sonication time:





5 s~30 mins





Ionic strength of





assembly





solution: no





requirement





At least one of





pre-processing





methods (such





as heating,





sonication or





quench): not





required


Mucous
General range: at
General
Ratio: 4.4~30 μg
siRNA,


membrane
least one dimension
range:
RNTs per
other



between 1 nm and
between +0 mV
0.1 nmol RNA
nucleic



150 nm
and
(Preferred ratio:
acids,



Preferred range: at
+60 mV
4.4~20 μg RNTs
molecular



least one dimension
Preferred
per 0.1 nmol
beacons and



between 10 nm and
range:
RNA)
peptides/



100 nm
between +0 mV
Sonication
proteins




and
power: 1~100%




+40 mV
(for a 700 W





sonicator)





Sonication time:





5 s~30 mins





Ionic strength of





assembly





solution: no





requirement





At least one of





pre-processing





methods (such





as heating,





sonication or





quench): not





required


Lung
General range: at
General
Ratio: 4.4~30 μg
siRNA,



least one dimension
range:
RNTs per
other



between 10 nm and
between +0 mV
0.1 nmol RNA
nucleic



150 nm
and
(Preferred ratio:
acids,



Preferred range: at
+60 mV
4.4~20 μg RNTs
molecular



least one dimension
Preferred
per 0.1 nmol
beacons and



between 20 nm and
range:
RNA)
peptides/



100 nm
between +0 mV
Sonication
proteins




and
power: 1~50%




+40 mV
(for a 700 W





sonicator)





Sonication time:





5 s~3 mins





Ionic strength of





assembly





solution: no





requirement





At least one of





pre-processing





methods (such





as heating,





sonication or





quench): not





required


Heart
General range: at
General
Ratio: 4.4~30 μg
siRNA,



least one dimension
range:
RNTs per
other



between 1 nm and 90 nm
between +0 mV
0.1 nmol RNA
nucleic



Preferred range: at
and
(Preferred ratio:
acids,



least one dimension
+60 mV
6.6~20 μg RNTs
molecular



between 1 nm and 30 nm
Preferred
per 0.1 nmol
beacons and




range:
RNA)
peptides/




between +8 mV
Sonication
proteins




and
power:




+40 mV
10%~100% (for





a 700 W





sonicator)





Sonication time:





10 s~30 mins





Ionic strength of





assembly





solution:





0~308 mmol/L





At least one of





pre-processing





methods (such





as heating,





sonication or





quench):





required









Diagnostic Applications

Molecular beacons or molecular beacon probes are oligonucleotide hybridization probes that report the presence of specific nucleic acids. Molecular beacons are hairpin shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid sequence. The use of molecular beacons is a non-radioactive method for detecting specific sequences of nucleic acids. They are useful in situations where it is either not possible or desirable to isolate the probe-target hybrids from an excess of the hybridization probes such as in the context of clinical diagnostics.
A typical molecular beacon probe is 25 nucleotides long. The middle 15 nucleotides are complementary to the target DNA or RNA and do not base pair with one another, while the five nucleotides at each terminus are complementary to each other rather than to the target DNA. A typical molecular beacon structure can be divided in 4 parts. Loop: a 18-30 base pair region of the molecular beacon that is complementary to the target sequence. Stem: the beacon stem is formed by the attachment, to both termini of the loop, of two short (5 to 7 nucleotide residues) oligonucleotides that are complementary to each other. 5′ fluorophore: located at the 5′ end of the molecular beacon, a fluorescent dye is covalently attached. 3′ quencher (non-fluorescent): the quencher dye part of the beacon is covalently attached to the 3′ end of the molecular beacon. When the beacon is in closed loop shape, the quencher resides in proximity to the fluorophore, which results in quenching the fluorescent emission of the latter.
If the nucleic acid to be detected is complementary to the strand in the loop, the event of hybridization occurs. The duplex formed between the nucleic acid and the loop is more stable than that of the stem because the former duplex involves more base pairs. This causes the separation of the stem and hence of the fluorophore and the quencher. Once the fluorophore is distanced from the quencher, illumination of the hybrid with light results in the fluorescent emission. The presence of the emission reports that the event of hybridization has occurred and hence the target nucleic acid sequence is present in the test sample. Molecular beacons are useful in SNP detection, real-time nucleic acid detection, real-time PCR quantification, allelic discrimination and identification, multiplex PCR assays, and for diaganostics. Nanopieces containing molecular beacons or other non-radioactive or radioactive detectable markers are particularly useful in diagnostic clinical assays.
MMP
MMP13 is involved in the progression of osteoarthritis. Matrix metalloproteinase (MMP) 13 is a major enzyme that targets cartilage for degradation. Compared to other MMPs, the expression of MMP13 is relatively more restricted to connective tissue. It not only targets type II collagen in cartilage for degradation, but also degrades proteoglycan, types IV and type IX collagen, osteonectin and perlecan in cartilage. Clinical investigation revealed that patients with articular cartilage destruction have high MMP13 expression, indicating that increased MMP13 is associated with cartilage degradation. MMP13-overexpressing transgenic mice developed a spontaneous OA-like articular cartilage destruction phenotype. The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of aggrecanases also contributes to proteoglycan/aggrecan depletion and are associated with cartilage degradation during OA. ADAMTS4 and 5 were identified as the major aggrecanases during OA development.
ADAMTS5
ADAMTS5 is a member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) protein family and a major aggrecanase in human cartilage. Members of the family share several distinct protein modules, including a propeptide region, a metalloproteinase domain, a disintegrin-like domain, and a thrombospondin type 1 (TS) motif. Individual members of this family differ in the number of C-terminal TS motifs, and some have unique C-terminal domains. The enzyme encoded by this gene contains two C-terminal TS motifs and functions as aggrecanase to cleave aggrecan, a major proteoglycan of cartilage.
ADAMTS5 plays a role in arthritis, e.g., it plays a key role in aggrecan degradation in cartilage. For example, genetically modified mice in which the catalytic domain of ADAMTS5 was deleted are resistant to cartilage destruction in an experimental model of osteoarthritis. ADAMTS5 is the major aggrecanase in mouse cartilage in a mouse model of inflammatory arthritis. ADAMTS5 is also useful as a biomarker for prediction of the response to infliximab (IFX) in patients with rheumatoid arthritis.
Fabrication of Tissue-Targeted Nanoparticles
Examples for the preparation of nanopieces for use in individual tissues are described below.
Cartilage/Chondrocytes:

    
    
        1) 30 μg RNTs in 50 μL water were heated to 99° C. for 3 mins, and then mixed with 0.1 nmol MMP-13 molecular beacon. The resulting mixture was sonicated at 100% power for 10 s.
        2) 4.4 μg RNTs in 1 μL water were sonicated at 50% power of a 700 W sonicator for 10 mins, and then mixed with 0.1 nmol miRNA-140. The resulting mixture was sonicated at 100% power for 30 mins.
        3) 10 μg RNTs in 10 μL water were sonicated at 100% power of a 700 W sonicator for 5 mins, and then mixed with 0.1 nmol ADAMTS-5 siRNA. The resulting mixture was sonicated at 100% power for 3 mins.
    
    


Synovium:

    
    
        1) 30 μg RNTs in 50 μL water were heated to 99° C. for 3 mins, and then mixed with 0.1 nmol IL-1β molecular beacon on ice. The resulting mixture was sonicated at 100% power for 60 s.
        2) 4.4 μg RNTs in 1 μL saline were sonicated at 1% power of a 700 W sonicator for 10 mins, and then mixed with 0.1 nmol IL-1 receptor antagonist protein. The resulting mixture was sonicated at 1% power for 10 s.
        3) 10 μg RNTs in 100 μL water were sonicated at 100% power of a 700 W sonicator for 5 mins, and then mixed with 0.1 nmol TNF-α siRNA. The resulting mixture was sonicated at 100% power for 60 s.
    
    


Neurons:

    
    
        1) 15 μg RNTs in 50 μL water at 1% power of a 700 W sonicator for 30 mins, and then mixed with 0.1 nmol VEGF molecular beacon. The resulting mixture was sonicated at 100% power for 60 s.
        2) 0.1 μg RNTs in 1 μL saline were heated to 99° C. for 3 mins, and then mixed with 0.1 nmol IL-1 receptor siRNA on ice. The resulting mixture was sonicated at 100% power for 30 mins.
        3) 10 μg RNTs were sonicated in 10 μL water at 100% power of a 700 W sonicator for 5 mins, and then mixed with 0.1 nmol MMP-1 siRNA. The resulting mixture was sonicated at 100% power for 3 mins.
    
    


Brain/BBB:

    
    
        1) 20 μg RNTs in 50 μL water were heated to 99° C. for 3 mins, and then mixed with 0.1 nmol MMP-9 molecular beacon on ice. The resulting mixture was sonicated at 100% power for 60 s.
        2) 1 μg RNTs in 1 μL saline were sonicated at 10% power of a 700 W sonicator for 10 mins, and then mixed with 0.1 nmol VEGF mRNA. The resulting mixture was sonicated at 10% power for 10 s.
        3) 10 μg RNTs in 10 μL water were sonicated at 100% power of a 700 W sonicator for 5 mins, and then mixed with 0.1 nmol TNF-α siRNA. The resulting mixture was sonicated at 100% power for 60 s.
    
    


Ocular Tissue:

    
    
        1) 30 μg RNTs in 50 μL water were heated to 99° C. for 3 mins, and then mixed with 0.1 nmol VEGF molecular beacon on ice. The resulting mixture was sonicated at 100% power for 60 s.
        2) 4.4 μg RNTs in 1 μL saline were sonicated at 1% power of a 700 W sonicator for 10 mins, and then mixed with 0.1 nmol VEGF antagonist protein. The resulting mixture was sonicated at 1% power for 10 s.
        3) 10 μg RNTs in 10 μL water were sonicated at 100% power of a 700 W sonicator for 5 mins, and then mixed with 0.1 nmol VEGF siRNA. The resulting mixture was sonicated at 100% power for 60 s.
    
    


Derm Tissue/Skin:

    
    
        1) 30 μg RNTs in 50 μL water were heated to 99° C. for 3 mins, and then mixed with 0.1 nmol IL-1β molecular beacon on ice. The resulting mixture was sonicated at 100% power for 60 s.
        2) 4.4 μg RNTs in 1 μL saline were sonicated at 1% power of a 700 W sonicator for 10 mins, and then mixed with 0.1 nmol IL-6 siRNA. The resulting mixture was sonicated at 1% power for 10 s.
        3) 10 μg RNTs in 10 μL water were sonicated at 100% power of a 700 W sonicator for 5 mins, and then mixed with 0.1 nmol IL-8 siRNA. The resulting mixture was sonicated at 100% power for 60 s.
    
    


Tumor:

    
    
        1) 30 μg RNTs in 50 μL water at 1% power of a 700 W sonicator for 30 mins, and then mixed with 0.1 nmol VEGF molecular beacon. The resulting mixture was sonicated at 100% power for 60 s.
        2) 0.1 μg RNTs in 1 μL saline were heated to 99° C. for 3 mins, and then mixed with 0.1 nmol TNF-α siRNA on ice. The resulting mixture was sonicated at 100% power for 30 mins.
        3) 10 μg RNTs were sonicated in 10 μL water at 100% power of a 700 W sonicator for 5 mins, and then mixed with 0.1 nmol MMP-1 siRNA. The resulting mixture was sonicated at 100% power for 3 mins.
    
    


Kidney:

    
    
        1) 30 μg RNTs in 50 μL water were heated to 99° C. for 3 mins, and then mixed with 0.1 nmol IL-12 molecular beacon on ice. The resulting mixture was sonicated at 100% power for 60 s.
        2) 4.4 μg RNTs in 1 μL saline were sonicated at 5% power of a 700 W sonicator for 10 mins, and then mixed with 0.1 nmol IL-1 receptor associated protein siRNA. The resulting mixture was sonicated at 1% power for 10 s.
        3) 10 μg RNTs in 10 μL water were sonicated at 100% power of a 700 W sonicator for 5 mins, and then mixed with 0.1 nmol IL-8 siRNA. The resulting mixture was sonicated at 100% power for 60 s.
    
    


Mucous Membrane:

    
    
        1) 30 μg RNTs in 50 μL water were heated to 99° C. for 3 mins, and then mixed with 0.1 nmol MMP-13 molecular beacon on ice. The resulting mixture was sonicated at 100% power for 60 s.
        2) 4.4 μg RNTs in 1 μL saline were sonicated at 1% power of a 700 W sonicator for 10 mins, and then mixed with 0.1 nmol MMP-9 siRNA. The resulting mixture was sonicated at 1% power for 10 s.
        3) 10 μg RNTs in 10 μL water were sonicated at 100% power of a 700 W sonicator for 5 mins, and then mixed with 0.1 nmol MMP-1 siRNA. The resulting mixture was sonicated at 100% power for 60 s.
    
    


Lung:

    
    
        1) 30 μg RNTs in 50 μL water were heated to 99° C. for 3 mins, and then mixed with 0.1 nmol TNF-α molecular beacon on ice. The resulting mixture was sonicated at 50% power for 60 s.
        2) 4.4 μg RNTs in 1 μL saline were sonicated at 1% power of a 700 W sonicator for 3 mins, and then mixed with 0.1 nmol MMP-9 siRNA. The resulting mixture was sonicated at 1% power for 5 s.
        3) 10 μg RNTs in 10 μL water were sonicated at 50% power of a 700 W sonicator for 1 mins, and then mixed with 0.1 nmol MMP-1 siRNA. The resulting mixture was sonicated at 100% power for 60 s.
    
    


Heart:

    
    
        1) 30 μg RNTs in 50 μL water were heated to 99° C. for 3 mins, and then mixed with 0.1 nmol VEGF molecular beacon. The resulting mixture was sonicated at 100% power for 10 s.
        2) 4.4 μg RNTs in 1 μL water were sonicated at 50% power of a 700 W sonicator for 10 mins, and then mixed with 0.1 nmol miRNA-365. The resulting mixture was sonicated at 100% power for 30 mins.
        3) 10 μg RNTs in 10 μL water were sonicated at 100% power of a 700 W sonicator for 5 mins, and then mixed with 0.1 nmol IL-1α siRNA. The resulting mixture was sonicated at 100% power for 3 mins.
    
    


Coating of Nanopieces, which is another important factor for tissue delivery can also be used to improve the tissue delivery. For example polyethylene glycol (PEG) and dextran are coatings often used.
The invention further provides methods for making composites of rosette nanotubes or components or rosette nanotubes or rosette Nanopieces and therapeutic or diagnostic agents including those known in the art. For example, agents include nucleic acids (DNA or RNA), wherein the RNA can be small RNA such as siRNA and miRNA. In particular, disclosed herein are novel siRNA transport complexes, comprising an unexpectedly advantageous transport vehicle. Methods of the present invention include contacting a transfection complex described herein with one or more cells, where the transfection complex includes a rosette nanotube and one or more nucleic acids such as DNA and RNA, for example siRNA. The rosette nanotube is a carrier that is formed from self-assembled modules as described below and those modules recognized in the art.
Compounds/Modules for Self-Assembly
Modules according to the present disclosure include compounds of Formula I below:




Wherein X is CH or nitrogen, preferably nitrogen; R2 is hydrogen or a linker group for example (CH2)n or other linker groups described herein, preferably (CH2)n; n is an integer of, 1, 2, 3, or 4, n=2 is preferred; Y is absent when R2 is hydrogen or is an amino acid or polypeptide having an amino group covalently bound to an α-carbon of the amino acid and the amino group is covalently bound to the linker group R2, Y is preferred to be lysine arginine, and histidine; and R1 is hydrogen or an aliphatic moiety, such as alkyl, straight or branched chain, saturated or unsaturated; and salts thereof. Preferably R1 is C1 to C10 alkyl, C1 to C5 alkyl, C1 to C3 alkyl, or methyl. Compounds within the scope of the invention include those where the Y group can be connected to the linker group either by the amino group or the carboxyl group of the amino acid or polypeptide. An exemplary linker group is shown in the formula below.




An exemplary module within the scope of Formula I is shown in FIG. 4 along with a schematic representation of a nanotube and an image of nanotubes formed from the exemplary module.
Alternative linker groups R2 can join the Y group to the carbon of the (CH2)n group or the N atom either by the amino group or the carboxyl group of the amino acid or polypeptide.
Alternative R2 groups within the scope of the present disclosure are selected from a group comprising:







wherein Y is absent.

Compounds of Formula I can be prepared by the methods described in U.S. Pat. No. 6,696,565 hereby incorporated by reference herein in its entirety alone or combined with methods known to those of skill in the art. Rosette nanotubes are made by assembly of compounds of Formula (I).
Exemplary compounds of Formula I are shown below:




Modules according to the present disclosure also include compounds of Formula II below:




Wherein X is CH or nitrogen preferably nitrogen; R2 is hydrogen or a linker group for example (CH2)n, preferably (CH2)n; where n is an integer of, 1, 2, 3, or 4 or (CH2)3CO or other linker groups described herein, n=2 is preferred; Y is absent when R2 is hydrogen or is an amino acid or polypeptide having an amino group covalently bound to an α-carbon of the amino acid and the amino group is covalently bound to the linker group R2, Y is preferred to be lysine arginine, and histidine; and R1 is hydrogen or an aliphatic moiety, such alkyl, straight or branched chain, saturated or unsaturated; and salts thereof. Preferably R1 is C1 to C10 alkyl, C1 to C5 alkyl, C1 to C3 alkyl, or methyl. An exemplary linker group is shown in the formula below.




Compounds within the scope of the present disclosure include those where the Y group can be connected to the linker group either by the amino group or the carboxyl group of the amino acid or polypeptide. Alternative R2 groups within the scope of the present disclosure are selected from a group comprising:







wherein Y is absent. TBL structures are made by the assembly of compounds of Formula (II).

Exemplary compounds of Formula II are shown below:










In some embodiments, compounds of formula II comprise amino acid functional group constructs. These compounds contain functional groups present in natural occurring amino acid side chains or may contain the entire amino acid side chain. For example, the lysine functional group construct contains the entire amino acid side chain functionality (—CH2CH2CH2CH2NH3+), whereas the histidine functional group construct only contains the heteroaryl imidazole group present in histidine.
In some embodiments, compounds of formula II comprise amino acid analogs. These compounds contain the entire the amino acid or may contain modified and/or unnatural amino acids. For example, the lysine amino acid analog contains the entire amino acid functionality of lysine, whereas the histidine amino acid analog contains a modified histidine amino acid.
In some embodiments the compounds of formula II are preferred over the compounds of formula I.
In some embodiments the compound of formula II is the Lysine Functional Group Construct:




According to certain aspects of the present disclosure, the structure of Formula II is referred to as a twin base with a linker (TBL) or twin base linkers insofar as two similar double ring structures are present as shown in Formula II and are linked to an amino acid or polypeptide. However, it is to be understood that the two double ring structures need not be identical insofar as they may have different X and R1 groups.
Embodiments of the present disclosure involve making composites of rosette nanotubes or components or rosette nanotubes or rosette Nanopieces and therapeutic or diagnostic agents including those known in the art and including nucleic acids, such as DNA or RNA. RNA can be small RNA including siRNA and miRNA. In particular, disclosed herein are novel siRNA transport complexes, comprising an unexpectedly advantageous transport vehicle. Methods of the present invention include contacting a transfection complex described herein with one or more cells, where the transfection complex includes a rosette nanotube and one or more nucleic acids such as DNA and RNA, for example siRNA. The rosette nanotube is a carrier that is formed from self-assembled modules as described below and those modules are recognized in the art.
TBL or twin base linkers comprise structures shown in Formula II and are linked to an amino acid, amino acid side chain structure, or polypeptide; compounds of Formula I may also be linked to an amino acid, amino acid side chain structure, or polypeptide. However, it is to be understood that the two double ring structures need not be identical insofar as they may have different X, Y, and R1 groups.
Amino acids can be divided into amino acid containing hydrophilic side chains, hydrophobic side chains, and electrically charged side chains. See chart below, wherein the side chains are shaded:
According to aspects of the present disclosure, modules (compounds) according to Formula I and Formula II self-assemble into substructures also called supermacrocycles which themselves will self-assemble into nanometer scale architectures or structures such as discrete nanotubular assemblies in water or aqueous solutions. Supermacrocycles are defined herein as being a number of organic molecules covalently or noncovalently bound together so as to form a ring structure. For example, compounds of Formula I will self-assemble into a 6-mer ring structure, sometimes referred to as a rosette. The process of forming nanotubes with the modules of the present disclosure is hierarchical. In particular, the modules of the present invention first self-assemble into supermacrocycles, and then the supermacrocycles self-assembly into nanotubes. Such self-assembly is described in U.S. Pat. No. 6,696,565. For the compounds of Formula II referred to as twin base linkers, the compounds will also assemble into a 6-mer ring structure. However, a single supermacrocycle formed will include two base layers owing to the presence of the two bases in each of the compound of Formula II.
Examples of modules of the present disclosure comprise the compounds of Formula I and Formula II and may include low molecular weight synthetic DNA base analogues referred to by the nomenclature C^G (Fenniri et al, J. Am. Chem. Soc. 2001, 123, 3854-3855) and A^T. The C^G moiety, referred to as a single CG motif, possesses the Watson-Crick donor-donor-acceptor of guanine and the acceptor-acceptor-donor of cytosine and undergoes a self-assembly process, fueled by an array of hydrogen bonds, to produce a six-membered supermacrocycle or rosette. Stacking of these rosettes produced a nanotube of very high aspect ratio. Compounds within the scope of the present invention include a twin G^C motif denoted as (C^G)2. Like the single C^G motif, the twin C^G motif (C^G)2 also possesses the Watson-Crick donor-donor-acceptor of guanine and the acceptor-acceptor-donor of cytosine and undergoes a self-assembly process, fueled by an array of hydrogen bonds, to produce a six-membered supermacrocycle or ring structure (rosette) of twin configuration. Stacking of these twin rosettes produces a nanotube of very high aspect ratio and higher stability. Analogously, The A^T moiety, referred to as a single AT motif, also possesses the Watson-Crick donor-donor-acceptor of adenine and the acceptor-acceptor-donor of thymine and undergoes a self-assembly process as well, fueled by an array of hydrogen bonds, to produce a six-membered supermacrocycle or rosette. Stacking of these rosettes produces a nanotube of very high aspect ratio. Compounds within the scope of the present invention include a twin A^T motif denoted as (A^T)2. Like the single A^T motif, the twin A^T motif (A^T)2 also possesses the Watson-Crick donor-donor-acceptor of adenine and the acceptor-acceptor-donor of thymine and undergoes a self-assembly process, fueled by an array of hydrogen bonds, to produce a six-membered supermacrocycle or ring structure (rosette) of twin configuration. Stacking of these twin rosettes also produces a nanotube of very high aspect ratio and higher stability.
It should be understood that the above described Formula I and/or Formula II demonstrate that electrostatic, stacking and hydrophobic interactions can be effectively orchestrated by hydrogen bonds to direct the hierarchical assembly and organization of helical nanotubular architectures in an aqueous milieu. Helical nanotubular architectures within the scope of the present invention include those formed entirely from compounds of Formula I. Helical nanotubular architectures within the scope of the present invention include those formed entirely from compounds of Formula II. Further, helical nanotubular architectures within the scope of the present invention include those formed from one or more of the compounds of Formula I and one or more of the compounds of Formula II. For example, a supermacrocycle ring substructure having particular amino acid or polypeptide side chains formed from the compounds of Formula I can be stacked with a supermacrocycle ring substructure having particular amino acid or polypeptide side chains formed from compounds of Formula II. The rosette substructures formed from the compounds of Formula I and Formula II can be stacked in any desired sequence to form nanotubular structures of the present invention. Utilizing this aspect of the present invention, a wide variety of structurally different modules (e. g, compounds) can be synthesized and self-assembled into supermacrocycles and then nanotubular structures according to methods of the present invention.
Another aspect of the invention is the conversion of nanotubes to nanorods by altering pH, temperature, and usage of physical methods (e.g., sonication, heating and blending) to prepare different sizes of Nanopieces.
Before assembly with delivery cargo, length of nanotubes (based on either Formula I or II) range in size from 1 nm to 999 micron, e.g., 10 nm to 999 nm. Outer width of nanotubes range in size from 0.5 nm to 100 nm, e.g., 1 nm to 10 nm. Inner diameter of nanotubes range in size from 1 angstrom to 10 nm, e.g., 0.5 nm to 5 nm.
After assembly with delivery cargo, length of Nanopieces (based on either Formula I or II) range in size from 1 nm to 999 micron, e.g., 10 nm to 999 nm. Width of Nanopieces range in size from 1 nm to 999 nm, e.g., 10 nm to 100 nm.
Another aspect of the invention is the packaging of drug molecules, e.g., therapeutics and diagnostics, with nanotubes to alter their surface charge and more importantly process these nanotubes into Nanopieces of the right shape and size to penetrate tissue matrix. Therefore, it is not the drug molecules that are released from nanotubes that diffuse into tissue, it is the Nanopieces themselves that penetrate the tissue. Control of the surface charge of the Nanopieces is done via the ratio of delivery cargo and nanotubes and/or nanorods. A further aspect of the invention is the use of coatings for the Nanopieces for tissue delivery. For example, polyethylene glycol and/or dextran are coatings that when used can improve tissue delivery.
A further aspect of the invention is the delivery of cargo into cells. These drug molecules can be nucleic acid, peptides, proteins, aromatic small molecules or negatively charged small molecules.
In some embodiments, the prepared module of the invention has an overall yield of no less than 60%, e.g., no less than 70%, no less than 80%, or no less than 90%.
In some embodiments, the module of the method of the invention contains more than 80% of compound of Formula I or II. In some embodiments, the product of the method of the invention contains more than 85%, 90%, 92%, 95%, 97%, 98%, 98.5%, or 99% of compound of Formula I and/or II. For example, the product is free of undesired byproduct or starting material.
In some embodiments, the nanotube of the invention has an overall yield of no less than 60%, e.g., no less than 70%, no less than 80%, or no less than 90%.
In some embodiments, the nanotube of the method of the invention contains more than 80% of compound of Formula I or II. In some embodiments, the product of the method of the invention contains more than 85%, 90%, 92%, 95%, 97%, 98%, 98.5%, or 99% of compound of Formula I and/or II. For example, the product is free of undesired byproduct or starting material.
In some embodiments, the Nanopieces of the invention has an overall yield of no less than 60%, e.g., no less than 70%, no less than 80%, or no less than 90%.
In some embodiments, the Nanopieces of the method of the invention contains more than 80% of compound of Formula I or II. In some embodiments, the product of the method of the invention contains more than 85%, 90%, 92%, 95%, 97%, 98%, 98.5%, or 99% of compound of Formula I and/or II. For example, the product is free of undesired byproduct or starting material.
According to certain preferred aspects of the present invention, a nanotube is prepared from single base ring structures and twin base ring structures in any desired order. The nanotube can have one or more single base ring structures and one or more twin base ring structures. Likewise, a nanotube within the scope of the present invention can include a plurality of single base ring structures formed from compounds of Formula I and a plurality of twin base ring structures formed from compounds of Formula II stacked together, e.g. one next to the other via hydrogen bonding, to form the nanotube.
Nanotube-Agent Complexes
According to certain aspects, nucleic acids or polypeptides includes small RNA being a duplex of between about 10 to about 30 nucleic acids, between about 15 to about 25 nucleic acids and between about 20 to about 23 nucleic acids, and any values and ranges in between whether overlapping or not. The small RNA can be formed by one or more oligonucleotides. Small RNA includes RNA commonly referred to as interference RNA, dsRNA, ssRNA, saRNA, siRNA or miRNA or their derivatives, analogs, mimics and inhibitors. According to certain aspects, siRNA is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in the RNAi-related pathways. siRNA within the scope of the present disclosure includes double stranded RNA of about 21 nucleotides with a 2 nucleotide 3′ overhang on either end of the siRNA. Each siRNA strand has a 5′ phosphate group and a 3′ hydroxyl (—OH) group. The structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs. Particular exemplary sequences of siRNA are readily available to those of skill in the art through published literature and siRNA is commercially available from, for example, Qiagen. It is to be understood that the present disclosure is not to be limited to any particular siRNA sequence, but rather the present disclosure broadly describes the incorporation of siRNA into or with rosette nanotubes. One of skill in the art will readily recognize that all siRNA sequences, given the similar structure and function of covalently connected nucleotides, can be incorporated into or complexed with rosette nanotubes using the methods described herein and that an exhaustive listing of publicly known siRNA sequences need not be provided herein.
According to additional aspects, DNA includes any DNA desired to be expressed by a cell. DNA includes genes having known functions and expressing known proteins. Likewise, DNA suitable for transfecting a cell will be apparent to those of skill in the art of transfection and gene expression.
Manufacture and Use of Transfection Complexes
The present disclosure is directed to methods of forming a transfection complex, for example, by mixing one or more nucleic acids with fully formed rosette nanotubes or modules that self-assemble into rosette nanotubes, such as the compounds of Formula I or Formula II. According to one aspect, fully formed rosette nanotubes in the form of a powder is dissolved in water and heated to boiling. The solution is then cooled to room temperature. One or more nucleic acids in the form of a solution is then added to the solution of nanotubes at a suitable temperature and for a suitable period of time until a complex of the nanotube and one or more nucleic acids forms. Suitable ratios of the nucleic acid to nanotube include about 0.01:1 (wt/wt) to about 1:0.1 (wt/wt).
The invention is further directed to transfection complexes, which include small RNA, such as siRNA and a rosette nanotube. Transfection complexes in accordance with the present invention may include any of the rosette nanotubes of the present invention in combination with small RNA known to those of skill in the art.
According to certain aspects, cells within the scope of the present invention that can be transfected include osteoblasts, fibroblasts, stem cells, neuronal cells, connective tissue cells, keratinocytes, cardiac myocytes, chondrocytes, proteoglycans, synoviocytes, adipose, phagocytic, blood monocytes, mesenchymal stem cells, neural stem cells, islet cells, hepatocytes, smooth muscle cells, urothelial cells, neurons, Schwann cells, microgial cells, cancerous and non-cancerous cells, epithelial cells, endothelial cells, myofibroblasts, osteoclasts, macrophages, leukocytes, osteocytes, astrocytes etc. and the like. Additional cells include bacterial cells such as Staphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa, MRSA, E. coli, candida (yeast), Candida albicans, Streptococcus pneumoniae, Neisseria meningitides, Haemophilus influenzae, Streptococcus agalactiae, Listeria monocytogenes, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Mycobacterium, tuberculosis, Streptococcus pyogenes, Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Ureaplasma urealyticum, Haemophilus ducreyi, Helicobacter pylori, Campylobacter jejuni, Salmonella, Shigella, Clostridium, Enterobacteriaceae, Staphylococcus saprophyticus and the like. The above list is intended to be exemplary and not exhaustive. One of skill in the art will readily be able to identify additional cells within the scope of the present disclosure, which is directed to toward cells present in joints, tissue and/or organs.
In general, a cell to be transfected includes, but is not limited to, any animal, plant or bacterial cell that is susceptible to intracellular delivery of DNA or RNA such as siRNA using the transfection complex of the present invention either in vitro or in vivo. For example, cells from different species such as human, mouse, rat, pig, chicken, etc. may be used according to the present disclosure. Likewise, cells from different tissues or organs, such as cartilage (e.g, ear, nose, rib cage, bronchial tube, intervertebral disc, hyaline, fibrous, elastic), connective tissue (e.g. loose, dense, adipose, fibrous, elastic, lymphoid), conjunctive tissue, fibers (e.g., collagenous, elastic, reticular), synovium, neuronal tissue, muscle tissue, ligament, tendon, busae, fibroblast, beast cells, macrophages from the immune system, and astrocytes from the neuronal system may be used. Likewise, primary cells obtained directly from animals, plants or bacteria may be used and cell lines, such as commercially available immortalized cell, may be used. Likewise, normal cells may be used and diseased cells may be used, such as cancer cells. For example, suitable cellular targets include, without limitation, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes, various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and the like. In certain aspects, the cell is selected from the group consisting of synoviocytes, fibroblasts, monocytes, chondrocytes, collagen, endothelial cells, connective tissue cells, neuronal cells, muscle cells, hematopoietic stem cells and tumor cells.
According to certain embodiments, the cells include one or more cells selected from the group consisting of transformed, recombinant, malignant, and primary cell lines. It is believed that the rosette nanotubes of the present invention will be effective as carriers of DNA or RNA such as siRNA in most, if not all cell types and cell lines. Since complexes of the rosette nanotubes and nucleic acids are composed of covalently bound base pairs, one of skill would expect that such complexes will be universally recognized by all cell types for transfecting purposes.
Methods of transfecting cells in accordance with the present invention may also include forming the transfection complex by combining in aqueous media the modules of the rosette nanotube and one or more DNA sequences and/or one or more RNA sequences. The complex is allowed to form. Cells are then contacted with the complex. According to one aspect, one of skill in the art will recognize from the benefit of the present disclosure that doses, concentrations, ratios and conditions of RNT/nucleic acids incorporation can be within ranges. For example, between about 1 μL to about 100 μL, for example 10 μL, of 1 mg/mL RNTs can be mixed with about 1 μL to about 100 μL, for example 20 μL of 5 μM nucleic acids, such as siRNA, miRNA, nucleic acid probes or other nucleic acids, at a temperature of between about 0° C. to about 37° C. for between about 0.5 hours to about 48 hours and added into 1 mL cell culture medium for transfection. For example, the combination of RNT and nucleic acids can be maintained at 4° C. for 24 hours or can be maintained at room temperature for two hours. Mixing can be accomplished by simple mixing, mixing while heating to about 60° C. to about 100° C., sonication or other methods known to those of skill in the art. If heated, the combination may then be subjected to a temperature of between about 0° C. to about 37° C. for between about 0.5 hours to about 48 hours to result in formation or assembly of the nanotube/nucleic acid complex. For example, nanotubes can be modified to modulate the surface charge of the nanotubes comprising one or more DNA sequence and/or one or more RNA sequences by varying the RNT/nucleic acid ratio. A skilled person in the arts would recognize that cartilage, for example, is a negatively charged tissue matrix and nanotube carrying an overall positive charge would increase the residence time of such Nanopieces in cartilage tissue.
Method of Treatment
The present invention also provides methods of treating tissue, organ and/or joint disease comprising using the complexes or compositions of the present invention. In particular, methods are provided for treating a patient having a tissue, organ or joint disease, by administering to the patient a therapeutically effective amount of a complex or composition of the present invention. For in vivo therapies based on local injection (e.g., intra-articularly, intratumoral, and intramuscularly) the RNT/small RNA complex is advantageously water soluble and so may be administered as an aqueous injection.
According to aspects of the present disclosure, composites of rosette nanotubes and small RNA can be combined with a pharmaceutically acceptable agent and administered as a delivery composition to an individual for therapeutic purposes.
In accordance with certain examples, complexes of the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the complexes disclosed here and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Therapeutic Applications
Also encompassed are methods for treating a patient having a tissue, organ and/or joint disease, by administering to the patient cells that have been transfected by the methods disclosed herein. An aspect of an ex vivo delivery method of the present invention may include for example, (i) removing a cell from a subject; (ii) introducing siRNA into a cell by contacting the cell with a delivery composition (transfection complex or composition comprising such a transfection complex) comprising siRNA and a rosette nanotube; and (iii) reintroducing the cell into the subject. In addition, nanotubes having nucleic acids complexed therewith as described herein may be delivered in vivo to an individual in need of treatment where the nanotubes having nucleic acids complexed therewith enter cells within the individual and the nucleic acids regulate cellular expression of proteins. For example the nucleic acids may silence genes in a therapeutic manner to the extent that a protein is not expressed resulting in treatment or the nucleic acids may be expressed by the cell to produce proteins in a therapeutic manner resulting in treatment.
Examples of joint diseases (e.g. synovial, fibrous, cartilaginous) potentially treatable with the complex, compositions, and methods include, but are not limited to the following: autoimmune, degenerative, inflammatory, infectious, cancerous, viral, fungal, injured or trauma derived. These joint diseases may be the primary disease or may be caused by an existing disease and/or illness. Examples include polymyalgia rheumatica, rheumatoid arthritis, multiple sclerosis, Charcot's Joint, osteoarthritis, juvenile onset of rheumatoid arthritis (JRA), system lupus erythematosus (SLE), psoriatic arthritis, inflammatory bowel disease (IBS) arthritis, Whipple's disease, intestinal lipodystrupjy, ankylosing spondylitis (AS), reactive arthritis, Still's disease, avascular necrosis, bursitis, fibromyalgia, gout, hemochromatosis, hypothyroidism, lupus, Lyme disease, Fifths disease, osteomalacia, osteomyelitis, Paget's disease of bone, pseudogout, rickets, septic arthritis, tendinitis, diabetes, Ehlers-Danlos syndrome, costochondritis, Perthes' disease, Marfan syndrome, rheumatic fever, tubercular arthritis, pigmented villonodular synovitis, scleroderma, polymyositis, erythema nodosum, neuropathic arthropathy, sickle-cell disease, acromegaly, amyloidosis, acute crystal synovitis, pyogenic bacterial infection, scurvy, hemophilia, achondroplasia, herniation, diffuse iodophatic skeletal hyperostosis (DISH), ganglion, lumbar spinal stenosis, sacrolilac joint pain, SAPHO syndrome, polycythemia, Raynaud's phenomenon, hydroxyapatite, Behcet's syndrome, Felt's syndrome, hepatitis B, primary Sjoegrens, and polychondritis.
In another aspect of the invention, joint disease can also be the result of genetics, trauma (e.g., meniscus tears), mechanical injury (e.g., repetitive motion), nutrition deficiencies, and joint mal-alignment. Joints having suffered from an initial injury and/or trauma often develop joint disease over a period of time.
Examples of tissue diseases (e.g. epithelial, connective, muscle and nervous tissue) potentially treatable with the complex, compositions, and methods include, but are not limited to the following: autoimmune, degenerative, inflammatory, infectious, cancerous, viral, fungal, injured or trauma derived. These tissue and/or organ diseases may be the primary disease or may be caused by an existing disease and/or illness. Examples include amyloidosis, atrial fibrillation, convulsion, cramp, dermatomyositis, enchondroma, fibroma, lumbao, heritable connective tissue disorder (e.g., Marfan syndrome, Peyronie's disease, Ehlers-Danlos syndrome, Osteogenesis imperfecta, Stickler syndrome, Alport syndrome, Congenital contractural arachnodactyly), autoimmune connective tissue disorder (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis, Scleroderma, Sjoegren's syndrome, mixed connective tissue disease, psoriatic arthritis), scurvy, muscle disease (e.g., muscle tumour, muscular dystrophy, disuse atrophy, denervation atrophy, Duchenne muscular dystrophy, facioscapulohumoral muscular dystrophy), hepatic diseasemyasthenia gravis, myopathy, myositis, myositis ossificans, cancer, fibromyalgia, muscle fatigue, spasm, spasticity, sprain, strain, brain injury, spinal cord injury, gliomas, neuroeptheliomatous, hypertension, cardiovascular disease, diabetes, Alzheimer's disease, cystitis, AIDS, rickets, and nerve sheath tumors. Examples of tissues, organs and/or body systems affected by disease and may be treated with the compositions, and methods described therein, but are not limited to the following: Immune system, senory organs (e.g., organs of tase, smell, sight, hearing), digestive system (e.g., mouth, fauces, pharynx, esophagus, abdomen, stomach, small intestine, large intestine, liver, pancreas), urogenital apparatus, endocrinological systemt, metabolism, cardiovascular system (e.g., heart, blood pressure, arteries), hematology (e.g., blood chemistry), urinary organs (e.g., kidneys, ureters, urinary bladder, male urethra, female urethra, male genital organs (e.g., testes and their covering, ductus deferens, vesiculae seminales, ejaculatory ducts, penis, prostate, bulbourethral glands), female genital organs (e.g., ovaries, uterine tube, uterus, vagina, clitoris, Bartholin's glands, external organs, mammae)), ductless glands (e.g., thyroid, parathyroid, thymus, hypophysis cerebri, pineal body, chromaphil and corticol systems, spleen), reproduction, respiratory (e.g., larynx, trachea, bonchi, pleurae, mediastinum, lungs), central nervous system (e.g., nerves, nerve fibers), skin, epithelial (e.g., simple, stratified, pseudostratified columnar, glandular), connective (e.g., loose connective (e.g., areolar, adipose, reticular), and dense connective (e.g., dense regular, dense irregular)), cartilage (e.g., Hyaline, elastic, fibrous), muscle (e.g., skeletal muscle (e.g., type I, II, IIa, IIx, IIb), cardiac muscle, smooth muscle), nervous (e.g., neuron (e.g., motor neurons, interneuron, sensory neuron), neuroglia, spinal cord, nerves, brain).
In another aspect of the invention, cancers can also reside in the joint, tissue and/or organ either as a primary tumor (e.g., sarcoma, hemangiopericytoma, connective tissue neoplasm, chondroma, chondrosarcoma) or as a result of metastasis of a primary tumor at a different location in the body of the subject.
Ex vivo and in vivo gene therapy with siRNA can also be used in joint, tissue, and/or organ disease. These RNAi applications toward joint disease include, but are not limited to, 1) targeting proteins or enzymes relevant in the disease state; 2) targeting or reducing expression of factors that are relevant in the disease state; and 3) targeting genes to maintain or restore joint health and homeostasis. For example, genes of the current invention may include ADAMTS (e.g., ADAMTS-4, ADAMTS-5), MMPs (e.g., MMP-1, MMP-3, MMP-9, MMP-13 and other MMPs), ILs (e.g., IL-1α, IL-1β, IL-2, IL-6, IL-8, IL-12, IL-15, IL-20, IL-21 and other ILs), IL receptors, IL receptor associated proteins, IL receptor antagonists, HLA-DRB1, PADI4, PTPN22, TNFAIP3, megakaryocyte stimulating factor, osteoprotegerin, activator of NF-α ligand, STAT4, CCR6, TNFR-1, TNFR-2, RIP, TRADD, PAD2-PAD4, FOX3, CD-25, FAP, DPP, CD26, MK2, SIRT-1, FoxO3a, miR-24, miR-125-5p, muR-203, miR-140, miR-365, miR-146a, miR-27a, TNF-α, HLA, collagen type II, aggrecan, prostaglandins, immunoglobulins, IFN-γ, GM-CSF, PDGF, FGF, VEGF, BMPs (e.g., BMP-2, BMP-4, BMP-7, and other BMPs), TGF-β, IGF-1, IGF-2 and, their related receptor protein and the like. For example, the following genes or proteins may promote arthritis such as rheumatoid arthritis: ADAMTS, MMPs, ILs, IL receptors, IL receptor associated proteins, HLA, DRB1, PADI4 gene, PTPN22 gene, TNFAIP3 gene, STAT4 gene, TNFR-1, TNFR-2, RIP, TRADD, PAD2-PAD4 proteins, CCR6 gene, miR-24, miR-125a-5p, mIR-365 and miR-203. Genes and protein can also prevent arthritis such as Juvenile idiopathic arthritis: FOXP3 and CD-25. Moreover, genes and proteins and their receptors and combinations thereof can also inhibit arthritis such as rheumatoid arthritis or osteoarthritis: IL receptor antagonists, MK2, FAP, DPP-4/CD26, SIRT-1/FoxO3a, miR-140 and miR-27a. Lastly, genes and proteins and their receptors and combinations thereof can mediate arthritis progression and joint tissue regeneration (such as cartilage regeneration): FGF, VEGF, BMPs, TGF-β, IGF-1, IGF-2, miR-146a.
Nanopieces deliver siRNA, antisense and/or anti-microRNA to knockdown genes and their related proteins and protein receptors (e.g., ADAMTS, MMPs, IL-1). In another example, Nanopieces deliver miRNA and/or mRNA to increase the level of genes and their related proteins and protein receptors. For example, genes and expression their respective encoded proteins and/or corresponding protein receptors that promote arthritis or other joint diseases can be knocked down; while genes and expression of their encoded proteins and/or corresponding protein receptors that inhibit arthritis or other joint diseases can be increased. Gene expression and production of encoded proteins and/or corresponding protein receptors that mediate arthritis progression and joint tissue regeneration can be adjusted (either knocked down or increased) depending on the needs or clinical condition of the patient.
Ex vivo and in vivo gene therapy with siRNA could also be used in cancer of tissue and/or organs. These RNAi applications toward cancer include, but are not limited to, 1) reducing expression of growth factors, reducing proteins that augment the cell cycle (e.g., Raf-1, PI-3 kinase), growth factor receptors (e.g., EGFR, Her-2), or proteins critical for supporting cells of the tumor (e.g., VEGF, VEGFR1-2 for tumor endothelial cells); 2) targeting or reducing expression of factors that are anti-apoptotic (e.g., BCL-2); and 3) targeting proteins or enzymes that reduce immune activation toward tumor.
Cancers or neoplasms contemplated within the scope of the disclosure include, but are not limited to, carcinomas (i.e., malignant tumors derived from epithelial cells such as, for example, common forms of breast, prostate, lung and colon cancer), sarcomas (i.e., malignant tumors derived from connective tissue or mesenchymal cells), lymphomas (i.e., malignancies derived from hematopoietic cells), leukemias (i.e., malignancies derived from hematopoietic cells), germ cell tumors (i.e., tumors derived from totipotent cells. In adults most often found in the testicle or ovary; in fetuses, babies and young children, most often found on the body midline, particularly at the tip of the tailbone), blastic tumors (i.e., a typically malignant tumor which resembles an immature or embryonic tissue) and the like.
Examples of specific neoplasms intended to be encompassed by the present invention include, but are not limited to, acute lymphoblastic leukemia, myeloid leukemia, acute childhood myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma (e.g., cerebellar, cerebral), atypical teratoid/rhabdoid tumor, basal cell carcinoma, extrahepatic bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain tumor (e.g., brain stem glioma, central nervous system atypical teratoid/rhabdoid tumors, central nervous system embryonal tumors, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and/or pineoblastoma, visual pathway and/or hypothalamic glioma, brain and spinal cord tumors), breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor (e.g., gastrointestinal), carcinoma of unknown primary, central nervous system (e.g., atypical teratoid/rhabdoid tumor, embryonal tumors (e.g., lymphoma, primary), cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, central nervous system embryonal tumors, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing family of tumors, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), gallbladder cancer, gastric cancer, gastrointestinal tumor (e.g., carcinoid tumor, stromal tumor (gist), stromal cell tumor), germ cell tumor (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumor, glioma (e.g., brain stem, cerebral astrocytoma), hairy cell leukemia, head and neck cancer, hepatocellular cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell tumors, Kaposi sarcoma, kidney cancer, large cell tumors, laryngeal cancer (e.g., acute lymphoblastic, acute myeloid), leukemia (e.g., acute myeloid, chronic lymphocytic, chronic myelogenous, hairy cell), lip and/or oral cavity cancer, liver cancer, lung cancer (e.g., non-small cell, small cell), lymphoma (e.g., AIDS-related, Burkitt, cutaneous T cell, Hodgkin, non-Hodgkin, primary central nervous system), Waldenström macroglobulinemia, malignant fibrous histiocytoma of bone and/or osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, myelogenous leukemia (e.g., chronic, acute, multiple), chronic myeloproliferative disorders, nasal cavity and/or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and/or malignant fibrous histiocytoma of bone, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer (e.g., islet cell tumors), papillomatosis, paranasal sinus and/or nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumors of intermediate differentiation, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal, pelvis and/or ureter, transitional cell cancer, respiratory tract carcinoma involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Ewing family of tumors, Kaposi, soft tissue, uterine), Sézary syndrome, skin cancer (e.g., non-melanoma, melanoma, merkel cell), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with occult primary, metastatic, stomach cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma, cutaneous; testicular cancer, throat cancer; thymoma and/or thymic carcinoma, thyroid cancer, transitional cell cancer of the renal, pelvis and/or ureter; trophoblastic tumor, unknown primary site carcinoma, urethral cancer, uterine cancer, endometrial, uterine sarcoma, vaginal cancer, visual pathway and/or hypothalamic glioma, vulvar cancer, Waldenström macroglobulinemia, Wilms tumor and the like. For a review, see the National Cancer Institute's Worldwide Website (cancer.gov/cancertopics/alphalist). One of skill in the art will understand that this list is exemplary only and is not exhaustive, as one of skill in the art will readily be able to identify additional cancers and/or neoplasms based on the disclosure herein.
Examples of primary cancers as joint disease comprise connective tissue neoplasm, hemangiopericytoma, sarcoma, chondroma, chondrosarcoma, bone and the like.
Examples of genetic and/or non-neoplastic diseases potentially treatable with the complex, compositions, and methods include, but are not limited to the following: adenosine deaminase deficiency; purine nucleoside phosphorylase deficiency; chronic granulomatous disease with defective p47phox; sickle cell with HbS, β-thalassemia; Faconi's anemia; familial hypercholesterolemia; phenylketonuria; ornithine transcarbamylase deficiency; apolipoprotein E deficiency; hemophilia A and B; muscular dystrophy; cystic fibrosis; Parkinsons, retinitis pigmentosa, lysosomal storage disease (e.g., mucopolysaccharide type 1, Hunter, Hurler and Gaucher), diabetic retinopathy, human immunodeficiency virus disease virus infection, acquired anemia, cardiac and peripheral vascular disease, osteoporosis and arthritis. In some of these examples of diseases, the therapeutic gene may encode a replacement enzyme or protein of the genetic or acquired disease, an antisense or ribozyme molecule, a decoy molecule, or a suicide gene product.
Recombinant cells may be produced using the complexes of the present invention. Resulting recombinant cells can be delivered to a subject by various methods known in the art. In certain embodiments, the recombinant cells are injected, e.g., subcutaneously or intra-articular. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously or intra-articular. The cells can also be encapsulated in a suitable vehicle and then implanted in the subject (see, e.g., Dionne et al. PCT Publication WO92/19195, dated Nov. 12, 1992). The amount of cells administered depends on a variety of factors known in the art, for example, the desired effect, subject state, rate of expression of the chimeric polypeptides, etc., and can readily be determined by one skilled in the art.
Another aspect of the present disclosure provides methods of introducing a therapeutic or diagnostic agent into a cell or tissue matrix using rosette nanotubes. Biologically active agents also called “therapeutic agents” or “drugs” are complexed with rosette nanotubes to form nanotube-drug complex, which can enter the cell and/or tissue and release the drug. A person of skill in the art will recognize the drug as being compounds which include any synthetic or natural element or are compounds which when introduced into the body causes a desired biological response, such as altering body function. Non-limiting examples of drugs or biologically active agents or therapeutic agents include anti-inflammatory agents (e.g., steroidal and non-steroidal), analgesics, anesthetics, chemotherapeutic agents, anti-proliferative agents, cytotoxic agents, steroidal agents, antifungal agents, antiviral agents, immunosuppressive agents, and include small molecules. Further non-limiting examples of drugs or biologically active agents or therapeutic agents include peptides (such as RGD, KRSR, YIGSR, IKVAV and the like), aromatic bioactive molecules such as tamoxifen, dexamethasone, vitamin K and the like, antibiotics such as penicillin, streptomycin, gentamycin and the like, glucosamine, chondroitin, cortisone, glucocorticoids, hydrocortisone, hyaluronic acid, hydrocortisone, gentamycin and the like, and proteins such as bone morphogenetic proteins, matrillins and the like. Drugs or biologically active agents or therapeutic agents may be hydrophobic or hydrophilic. According to one aspect, the rosette nanotubes include hydrophobic moieties within the core portion of the structure where hydrophobic drugs, biologically active agents or therapeutic agents may be located in the composite. According to another aspect, the rosette nanotubes of the present disclosure may have hydrophilic outer surfaces to facilitate administration of the complexes in physiological environments.
Examples of analgesic agents include opioid analgesics and adjuvant analgesics within the scope of the present disclosure that can be complexed with rosette nanotubes include clonidine, tizanidine, gapapentin, pregabalin, lamotrigine, oxcarbazepine, topiramate, levitiracetam, tigabine, zonisamide, carbamazepine, valprioc acid, phenytoin, amitriptyline, nortriptyline, desipramine, imipramine, doxepin, paroxetine, citalopram, escitalopram, fluoxetine, venlafaxine, duloxetine, bupriopion, mexiletine, lidocaine, baclofen, cyclobenzaprine, orphenadrine, metaxalone, methocarbamol, morphine, hydrocodone, hydromorphone, tramadol, oxycodone, oxymorphone, fentanyl, methadone, capsaicin, loperamide, naloxone, demerol, buprenorphine, butorphanol, codeine, levorphanol, meperidine, methadone, nabuphine, propoxyphene, and pentazocine.
Examples of non-opioid and anti-inflammatory agents within the scope of the present disclosure that can be complexed with rosette nanotubes include acetaminophen, aspirin, diflunisal, choline magnesium trisalicylate, salsalate, ibuprofen, naproxen, ketoprofen, fluriprofen, oxaprozin, indomethacin, sulindac, nabumetone, diclofenac, ketorolac, tolectin, piroxicam, meloxicam, mefenamic acid, meclofenamate, celecoxib, allopurinol, dextromethorphan, pegloticase, dexibuprofen, etodolac, fenoprofen, flufenamic acid, flupbiprofen, lornoxicam, loxoprofen, meclofenamic acid, piroxicam, tenoxicam, tolmetin, and tolfenamic acid.
Examples of immunosuppresive agents within the scope of the present disclosure that can be complexed with rosette nanotubes include alkylating agents, antimetabolites, high dose corticosteroids, azathioprine, mycophenolate mofetil, cyclosporine, methotrexate, leflunomide, cyclophosphamide, chlorambucil, nitrogen mustard, abacavir, abciximab, adalimumab, aldesleukin, altretamine, aminoglutethimide, amprevenir, anakinra, anastrozole, aspariginase, azathioprine, basiliximab, betamethasone, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cidofovir, cisplatin, cladribine, cortisone, cyclosporine, cytarabine, decarbazine, dacuzumab, dactinomycin, daunorubicin, delaviridine, dexamethasone, didanosine, doxorubicin, efavirenz, epirubicin, estramustine, etanercept, etoposide, exemestane, foxuridine, fludarabine, fluorouracil, flutamide, gemcitabine, gemtuzumab ozogamicin, hydrocortisone, hydroxychloroquine, hydroxyurea, idaubicin, ifosphamide, indinavir, infliximab, interferon alpha-2a, interferon alpha-2b, interferon beta-2b, interferon beta-2a, interferon gamma-1b, interleukin-2, irinotecan, isotretinoin, lamivudine, leflunomide, letrozole, leuprolide, mechloethamine, megestrol, melphalan, mercaptopurine, methotrexate, methylpregnisolone, mitomycin, mitotane, mitoxantrone, mycophenolate, nelfinavir, nevirapine, paclitaxel, pegaspargase, penicillamine, pentostatin, pimecroslimus, pipobroman, plicamycin, prednisolone, predisone, priliximab, procarbazine, ritonavir, rituximab, saquinavir, sargamomstim, stavudine, strepozocin, tacrolismus, temozolomide, teniposide, testolactone, thioguanine, thiotepa, trastuzumab, tretinoin, triamcinolone, uracil mustard, valrubucin, vinblastine, vincristine, vinorelbine, zalcitabine, zidovudine.
Examples of antifungal agents within the scope of the present disclosure that can be complexed with rosette nanotubes include polyene, azole, allylamine, morpholine, and antimetabolite antifungal agents, e.g., amphotericin B, candicin, filipin, hamycin, natamycin, nystatin rimocidin, bifonazole, butoconazole, clotrimazole, econozole, fenticonazole, isoconazole, ketoconazole, luiconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, fluconazole, isavuconazole, traconazole, posaconazole, ravuconazole, terconazole, voriconazole, abafungin, amorolfin, butenafine, naftifine, terbinafine, anidulafungin, caspofungin, micafungin, benzoic acid, ciclopirox, griseofulvin, tolnaftate, and undecylenic acid.
Examples of antibiotic agent within the scope of the present disclosure that can be complexed with rosette nanotubes include aminoglycosides (e.g., amikacin, gentamicin, kanamycine, neomycine, metilmicin, tobramycin, paromomycin, streptomycin, spectinomycin), anasamycins (e.g., geldanamycin, herbimycin, riflaximin), loracerbef, carbapenems (e.g., ertapenem, doripenem, cilastatin, meropenem), cephalosporin (e.g. cefadroxil, cefazolin, cephalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefdotoren, cefotaxime, ceftibuten, ceftizoxime, cefepime, ceftaroline, ceftobioprole, teichoplanin, vancomycin, telavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spiramycin, azetreonam, flurazolidone, linezolid, posizolid, radezolid, torezolid, ampicillin, azolocillin, carbenicillin, cloxacillin, dicloxaxillin, pencillin), polypeptides (e.g. bacitracin, colistin, polymyxin B), Quinolones (e.g., ciproflaxin, enoxacin, gemifloxacin, norfloxacin), sulfonamides (e.g., malfenide, sulfamethizole, sulfasalazine, sulfadiazine), tetracyclines (e.g., demeclocycline, minocycline, doxycycline, tetracycline), clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, riflampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramthenicol, foffmycin, fusidic acid, metronidazole, mupirocin, platensimycin, thiamphenicol, tigecycline, tinidazole, and trimethoprim.
Examples of drugs within the scope of the present disclosure that can be complexed with rosette nanotubes include glucosamine, chondroitin, cortisone, glucocorticoids, hydrocortisone, hyaluronic acid, hydrocortisone, and lurbicants (e.g. lubricin).
Examples of anti-cancer drugs within the scope of the present disclosure that can be complexed with rosette nanotubes include bortezomib ([(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl) amino]propyl]amino]butyl] boronic acid; MG-341; VELCADE®), MG-132 (N-[(phenylmethoxy)carbonyl]-L-leucyl N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide); pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine); purine analogs; folate antagonists and related inhibitors (e.g., mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine [cladribine]); folic acid analogs (e.g., methotrexate); antimitotic agents, including vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine) and alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (e.g., carmustine (BCNU) and analogs, streptozocin), trazenes—dacarbazinine (DTIC); microtubule disruptors (e.g., paclitaxel, docetaxel, vincristin, vinblastin, nocodazole, epothilones and navelbine, and teniposide); actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosourea, paclitaxel, plicamycin, procarbazine, teniposide, triethylenethiophosphoramide and etoposide (VP 16); dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; L-asparaginase; antiplatelet agents; platinum coordination complexes (e.g., cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones and hormone analogs (e.g., estrogen, tamoxifen, goserelin, bicalutamide, nilutamide); aromatase inhibitors (e.g., letrozole, anastrozole); anticoagulants (e.g., heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, COX-2 inhibitors, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (e.g., breveldin); immunosuppressives (e.g., cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (e.g., TNP-470, genistein) and growth factor inhibitors (e.g., vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors, epidermal growth factor (EGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; anti-sense oligonucleotides; antibodies (e.g., trastuzumab (HERCEPTIN®), AVASTIN®, ERBITUX®); cell cycle inhibitors and differentiation inducers (e.g., tretinoin); mTOR (mammalian target of rapamycin) inhibitors (e.g., everolimus, sirolimus); topoisomerase inhibitors e.g., doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan); corticosteroids (e.g., cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers; and caspase activators and the like.
Examples of anti-cancer drugs within the scope of the present disclosure that can be complexed with rosette nanotubes include alemtuzumab; aminoglutethimide; amsacrine; anastrozole; asparaginase; bevacizumab; bicalutamide; bleomycin; bortezomib; buserelin; busulfan; campothecin; capecitabine; carboplatin; carmustine; CeaVac; cetuximab; chlorambucil; cisplatin; cladribine; clodronate; colchicine; cyclophosphamide; cyproterone; cytarabine; dacarbazine; daclizumab; dactinomycin; daunorubicin; dienestrol; diethylstilbestrol; docetaxel; doxorubicin; edrecolomab; epirubicin; epratuzumab; erlotinib; estradiol; estramustine; etoposide; exemestane; filgrastim; fludarabine; fludrocortisone; fluorouracil; fluoxymesterone; flutamide; gemcitabine; gemtuzumab; genistein; goserelin; huJ591; hydroxyurea; ibritumomab; idarubicin; ifosfamide; IGN-101; imatinib; interferon; irinotecan; ironotecan; letrozole; leucovorin; leuprolide; levamisole; lintuzumab; lomustine; MDX-210; mechlorethamine; medroxyprogesterone; megestrol; melphalan; mercaptopurine; mesna; methotrexate; mitomycin; mitotane; mitoxantrone; mitumomab; nilutamide; nocodazole; octreotide; oxaliplatin; paclitaxel; pamidronate; pentostatin; pertuzumab; plicamycin; porfimer; procarbazine; raltitrexed; rituximab; streptozocin; sunitinib; suramin; tamoxifen; temozolomide; teniposide; testosterone; thalidomide; thioguanine; thiotepa; titanocene dichloride; topotecan; tositumomab; trastuzumab; tretinoin; vatalanib; vinblastine; vincristine; vindesine; and vinorelbine and the like.
Examples of NMDA receptor antagonists within the scope of the present disclosure that can be complexed with rosette nanotubes include LY 274614 (decahydro-6-(phosphonomethyl)-3-isoquinolinecarboxylic acid), LY 235959 [(3S,4aR,6S,8aR)-decahydro-6-(phosphonomethyl)-3-isoquinolinecarboxylic acid], LY 233053 ((2R,4S)-rel-4-(1H-tetrazol-5-yl-methyl)-2-piperidine carboxylic acid), NPC 12626 (α-amino-2-(2-phosphonoethyl)-cyclohexanepropanoic acid), reduced and oxidized glutathione, carbamathione, AP-5 (5-phosphono-norvaline), CPP (4-(3-phosphonopropyl)-2-piperazinecarboxylic acid), CGS-19755 (seifotel, cis-4(phono-methyl)-2-piperidine-carboxylic acid), CGP-37849 ((3E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid), CGP 39551 ((3E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid, 1-ethyl ester), SDZ 220-581 [(αS)-α-amino-2′-chloro-5-(phosphonomethyl)-[1,1′-biphenyl]-3-propanoic acid], and S-nitrosoglutathione. amantadine, aptiganel (CERESTAT®, CNS 1102), caroverine, dextrorphan, dextromethorphan, fullerenes, ibogaine, ketamine, lidocaine, memantine, dizocilpine (MK-801), neramexane (MRZ 2/579, 1,3,3,5,5-pentamethyl-cyclohexanamine), NPS 1506 (delucemine, 3-fluoro-γ-(3-fluorophenyl)-N-methyl-benzenepropanamine hydrochloride), phencyclidine, tiletamine and remacemide. acamprosate, arcaine, conantokin-G, eliprodil (SL 82-0715), haloperidol, ifenprodil, traxoprodil (CP-101,606), and Ro 25-6981 [(±)-(R,S)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidine propanol]; aminocyclopropanecarboxylic acid (ACPC), 7-chlorokynurenic acid, D-cycloserine, gavestinel (GV-150526), GV-196771 A (4,6-dichloro-3-[(E)-(2-oxo-1-phenyl-3-pyrrolidinylidene)methyl]-1H-indole-2-carboxylic acid monosodium salt), licostinel (ACEA 1021), MRZ-2/576 (8-chloro-2,3-dihydropyridazino[4,5-b]quinoline-1,4-dione 5-oxide 2-hydroxy-N,N,N-trimethyl-ethanaminium salt), L-701,324 (7-chloro-4-hydroxy-3-(3-phenoxyphenyl)-2(1H)-quinolinone), HA-966 (3-amino-1-hydroxy-2-pyrrolidinone), and ZD-9379 (7-chloro-4-hydroxy-2-(4-methoxy-2-methylphenyl)-1,2,5,10-tetra-hydropyridanizo[4,5-b]quinoline-1,10-dione, sodium salt); oxidized and reduced glutathione, S-nitrosoglutathione, sodium nitroprusside, ebselen, and disulfiram, DETC-MeSO, carbamathione; CNQX (1,2,3,4-tetrahydro-7-nitro-2,3-dioxo-6-quinoxalinecarbonitrile) and DNQX (1,4-dihydro-6,7-dinitro-2,3-quinoxalinedione) and the like.
Examples of subtype-specific NMDA receptor antagonists within the scope of the present disclosure that can be complexed with rosette nanotubes include arcaine, argiotoxin636, Co 101244 (PD 174494, Ro 63-1908, 1-[2-(4-hydroxyphenoxy)ethyl]-4-[(4-methylphenyl)methyl-4-piperidinol], despiramine, dextromethorphan, dextrorphan, eliprodil, haloperidol, ifenprodil, memantine, philanthotoxin343, Ro-25-6981 ([(±)-(R*,S*)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidine propanol]), traxoprodil (CP-101,606), Ro 04-5595 (1-[2-(4-chlorophenyl)ethyl]-1,2,3,4-tetrahydro-6-methoxy-2-methyl-7-isoquinolinol), CPP [4-(3-phosphonopropyl)-2-piperazinecarboxylic acid], conantokin G, spermine, spermidine, NVP-AAM077 [[[[(1S)-1-(4-bromophenyl)ethyl]amino](1,2,3,4-tetrahydro-2,3-dioxo-5-quinoxalinyl)methyl]-phosphonic acid]; and 1-(phenanthrene-2-carbonyl) piperazine-2,3-dicarboxylic acid and the like.
Examples of anticonvulsants within the scope of the present disclosure that can be complexed with rosette nanotubes include barbiturates (e.g., mephobarbital and sodium pentobarbital); benzodiazepines, such as alprazolam (XANAX®), lorazepam, clonazepam, clorazepate dipotassium, and diazepam (VALIUM®); GABA analogs, such as tiagabine, gabapentin (an α2δ antagonist, NEURONTIN®), and β-hydroxypropionic acid; hydantoins, such as 5,5-diphenyl-2,4-imidazolidinedione (phenytoin, DILANTIN®) and fosphenytoin sodium; phenyltriazines, such as lamotrigine; succinimides, such as methsuximide and ethosuximide; 5H-dibenzazepine-5-carboxamide (carbamazepine); oxcarbazepine; divalproex sodium; felbamate, levetiracetam, primidone; zonisamide; topiramate; and sodium valproate.
Examples of psychiatric drugs within the scope of the present disclosure that can be complexed with rosette nanotubes include Abilify, Adapin, Adartrel, Adderall, Alepam, Alertec, Aloperidin, Alplax, Alprax, Alprazolam, Alviz, Alzolam, Amantadine, Ambien, Amisulpride, Amitriptyline, Amoxapine, Amfebutamone, Anafranil, Anatensol, Ansial, Ansiced, Antahus, Antabuse, Antideprin, Anxiron, Apo-Alpraz, Apo-Primidone, Apo-Sertral, Aponal, Apozepam, Aripiprazole, Aropax, Artane, Asendin, Asendis, Asentra, Ativan, Atomoxetine, Aurorix, Aventyl, Axoren, Beneficat, Benperidol, Bimaran, Bioperidolo, Biston, Brotopon, Bespar, Bupropion, Buspar, Buspimen, Buspinol, Buspirone, Buspisal, Cabaser, Cabergoline, Calepsin, Calcium carbonate, Calcium carbimide, Calmax, Carbamazepine, Carbatrol, Carbolith, Celexa, Chloraldurat, Chloralhydrat, Chlordiazepoxide, Chlorpromazine, Cibalith-S, Cipralex, Citalopram, Clomipramine, Clonazepam, Clozapine, Clozaril, Concerta, Constan, Convulex, Cylert, Dapotum, Daquiran, Daytrana, Defanyl, Dalmane, Damixane, Demolox, Depad, Depakene, Depakote, Depixol, Desyrel, Dostinex, dextroamphetamine, Dexedrine, Diazepam, Didrex, Divalproex, Dogmatyl, Dolophine, Droperidol, Edronax, Efectin, Effexor (Efexor), Eglonyl, Einalon S, Elavil, Elontril, Endep, Epanutin, Epitol, Equetro, Escitalopram, Eskalith, Eskazinyl, Eskazine, Etrafon, Eukystol, Eunerpan, Faverin, Fazaclo, Fevarin, Finlepsin, Fludecate, Flunanthate, Fluoxetine, Fluphenazine, Flurazepam, Fluspi, Fluspirilen, Fluvoxamine, Focalin, Gabapentin, Geodon, Gladem, Glianimon, Halcion, Halomonth, Haldol, Haloperidol, Halosten, Imap, Imipramine, Imovane, JJanimine, Jatroneural, Kalma, Keselan, Klonopin, Lamotrigine, Largactil, Lecital, Levomepromazine, Levoprome, Leponex, Lexapro, Libritabs, Librium, Linton, Liskantin, Lithane, Lithium, Lithizine, Lithobid, Lithonate, Lithotabs, Lorazepam, Loxapac, Loxapine, Loxitane, Ludiomil, Lunesta, Lustral, Luvox, Lyrica, Lyogen, Manegan, Manerix, Maprotiline, Mellaril, Melleretten, Melleril, Melneurin, Melperone, Meresa, Mesoridazine, Metadate, Methamphetamine, Methotrimeprazine, Methylin, Methylphenidate, Minitran, Mirapex, Mirapexine, Moclobemide, Modafinil, Modalina, Modecate, Moditen, Molipaxin, Moxadil, Murelax, Myidone, Mylepsinum, Mysoline, Nardil, Narol, Navane, Nefazodone, Neoperidol, Neurontin, Nipolept, Norehox, Normi son, Norpramine, Nortriptyline, Novodorm, Olanzapine, Omca, Oprymea, Orap, Oxazepam, Pamelor, Parnate, Paroxetine, Paxil, Peluces, Pemoline, Pergolide, Permax, Permitil, Perphenazine, Pertofrane, Phenelzine, Phenytoin, Pimozide, Piportil, Pipotiazine, Pragmarel, Pramipexole, Pregabalin, Primidone, Prolift, Prolixin, Promethazine, Prothipendyl, Protriptyline, Provigil, Prozac, Prysoline, Psymion, Quetiapine, Ralozam, Reboxetine, Resimatil, Restoril, Restyl, Requip, Rhotrimine, Risperdal, Risperidone, Rispolept, Ritalin, Rivotril, Ropark, Ropinerole, Rubifen, Rozerem, Sediten, Seduxen, Selecten, Serax, Serenace, Serepax, Serenase, Serentil, Seresta, Serlain, Serlift, Seroquel, Seroxat, Sertan, Sertraline, Serzone, Sevinol, Sideril, Sifrol, Sigaperidol, Sinequan, Sinqualone, Sinquan, Sirtal, Solanax, Solian, Solvex, Songar, Stazepin, Stelazine, Stilnox, Stimuloton, Strattera, Sulpiride, Sulpiride Ratiopharm, Sulpiride Neurazpharm, Surmontil, Symbyax, Symmetrel, Tafil, Tavor, Taxagon, Tegretol, Telesmin, Temazepam, Temesta, Temposil, Terfluzine, Thioridazine, Thiothixene, Thombran, Thorazine, Timonil, Tofranil, Tradon, Tramadol, Tramal, Trancin, Tranax, Trankimazin, Tranquinal, Tranylcypromine, Trazalon, Trazodone, Trazonil, Trialodine, Trevilor, Triazolam, Trifluoperazine, Trihexane, Trihexyphenidyl, Trilafon, Trimipramine, Triptil, Trittico, Troxal, Tryptanol, Ultram, Valium, Valproate, Valproic acid, Valrelease, Vasiprax, Venlafaxine, Vestra, Vigicer, Vivactil, Wellbutrin, Xanax, Xanor, Xydep, Zamhexal, Zeldox, Zimovane, Zispin, Ziprasidone, Zolarem, Zoldac, Zoloft, Zolpidem, Zonalon, Zopiclone, Zotepine, Zydis, Zyprexa and the like.
Examples of miscellaneous drugs within the scope of the present disclosure that can be complexed with rosette nanotubes include nortriptyline, amytriptyline, fluoxetine (PROZAC®), paroxetine HCl (PAXIL®), trimipramine, oxcarbazepine (TRILEPTAL®), eperisone, misoprostol (a prostaglandin E1 analog), latanoprost (a prostaglandin F2  analog) melatonin, and steroids (e.g., pregnenolone, triamcinolone acetonide, methylprednisolone, and other anti-inflammatory steroids) and the like.
Examples of antiviral drugs within the scope of the present disclosure that can be complexed with rosette nanotubes include Abacavir, Aciclovir, Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla (fixed dose drug), Boceprevir, Cidofovir, Combivir (fixed dose drug), Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Entry inhibitors, Famciclovir, Fixed dose combination (antiretroviral), Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet, Fusion inhibitor, Ganciclovir, Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir, Inosine, Integrase inhibitor, Interferon type III, Interferon type II, Interferon type I, Interferon, Lamivudine, Lopinavir, Loviride, Maraviroc, Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir, Nucleoside analogues, Oseltamivir (Tamiflu), Peginterferon alfa-2a, Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, Protease inhibitor (pharmacology), Raltegravir, Reverse transcriptase inhibitor, Ribavirin, Rimantadine, Ritonavir, Pyramidine, Saquinavir, Stavudine, Synergistic enhancer (antiretroviral), Tea tree oil, Tenofovir, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir (Valtrex), Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir (Relenza), Zidovudine and the like.
Ex vivo and in vivo therapy and/or diagnostics could also be used in joint disease. These therapeutic and diagnostic applications toward these joint diseases include, but are not limited to, 1) targeting proteins or enzymes relevant in the disease state; 2) targeting or reducing expression of factors that are relevant in the disease state; and 3) targeting genes to maintain or restore joint health and homeostasis. For example, Nanopieces delivery of molecular probes to detect expression of inflammatory markers (e.g., cytokines, MMP, ADAMS) and the like or delivery of therapeutic agents to treat pain, inflammation, infection and the like can be used.
In another example, in vivo imaging technology to detect molecular changes at early stages of arthritis without harming articular cartilage was demonstrated. Osteoarthritis (OA) is one of the most common causes of disability. However, the lack of tools for early diagnosis of OA hampers the prevention and treatment of the disease to decelerate articular cartilage loss and alleviate suffering of patients. The OA Biomarker Initiative has identified a series of biomarkers, including Matrix metalloproteinases (MMP), which are elevated in articular cartilage during OA pathogenesis. However, detection of MMP protein levels or activities in serum may not be sensitive enough, while the more sensitive detection of MMP transcripts requires invasive procedure to obtain biopsy of articular joint tissue. Therefore, there is an urgent need to develop sensitive in vivo imaging technology to detect molecular changes at early stages of arthritis without harming articular cartilage.
Specifically, Molecular beacon (MB) technology provided an intriguing possibility to detect the changes of mRNA levels in live animals in vivo. In fact, molecular beacon (MB) technology (FIG. 51) detected the changes of mRNA levels in live animals in vivo. The Molecular beacon comprises an oligonucleotides loop, double strand stem, and a fluorophore and quencher, which remains non-fluorescent due to the proximity of fluorophore and quencher. Upon entering a cell and hybridizing with its target mRNA, MB emits fluorescence after separation of the fluorophore and quencher (FIG. 52). However, prior to the invention, there was no report of detection of OA using MB due to the significant challenge of in vivo delivery of MB into joint tissues. Detection of OA using MB is challenging because of the in vivo delivery of MB into joint tissues. Early detection of OA in the Destabilizing Medial Meniscus (DMM) mouse OA model using MB to detect induction of MMP-13 transcript, a major matrix proteinase that degrades interstitial collagen matrix during arthritis was shown. In vivo delivery of MMP13 MB using Nanopieces derived from rosette nanotubes were used. Since cartilage is a very negatively charged tissue (containing a huge amount of proteoglycan), the negatively charged Nanopieces intend to bind and accumulate onto and/or into the matrix and/or tissue resulting in much longer retention time to achieve more effective delivery. Different sizes of Nanopieces can be created for different delivery proposes to get into the matrix. For example, cartilage tissue matrix has about 60 nm mesh size of the collagen II fibrillar network and about 20 nm spacing between the side chains of the proteoglycan network. Nanopieces with small sizes (at least one dimension smaller than 60 nm and/or 20 nm) showed excellent efficiency and function in intra-cartilage matrix delivery of siRNA. Adjusting the ratio between RNTs and cargo reagents to yield an overall positive charged surface enabled Nanopieces to adhere with negatively charged matrix and/or tissue components resulting longer retention time.
Intra-joint delivery was thereby achieved with these processed Nanopieces. Delivery of Molecular probes with Nanopiece detected a specific gene expression (or protein activity) along with the co-delivery of a negative control for non-specific signal and an internal positive control to accurately diagnose a target gene expression in a real-time, in-situ and non-invasive manner. Matrix metalloproteinases (MMP) are the major enzymes that degrade the components of the extracellular matrix during arthritis progression. MMP-13, which is usually produced by cartilage and bone, degrade interstitial collagens (types I, II and III) in both OA and RA. Expression of MMP-13 is low in normal cells, whereas in pathologic condition excess MMP-13 production is associated with inflammation. mRNA level of MMP-13 are indicative for arthritis development and MMP-13 is as a good target in early diagnosis of arthritis. However, articular cartilage tissues need to be collected to show the up-regulation of MMP-13 mRNA levels. The combination of molecular beacon and Nanopieces technology detected of OA in vivo in a specific and sensitive manner without harming any joint tissues.
In another example, therapeutic agents complexed with nanotubes can knock down one or multiple disease gene expression (such as via siRNA delivery) and/or up-regulate one or multiple beneficial gene and/or protein (such as via DNA, mRNA or protein delivery) and deliver a variety of cargo types and can deliver multiple cargo reagents at the same time.
Accordingly, the rosette nanotubes of the present disclosure have hollow channels that can be used for drug encapsulation. Rosette nanotubes are able to incorporate water-insoluble drugs into their tubular structures by hydrophobic interactions with the core whereas their hydrophilic outer surface can shield such hydrophobic drugs in a physiological environment for subsequent prolonged release (even into the cell). Rosette nanotubes can also be chemically functionalized with peptides such as Arg-Gly-Asp-Ser-Lys, Lys-Arg-Ser-Arg-Lys, and Gly-Arg-Gly-Asp-Tyr-Lys to deliver growth factors for healthy tissue regeneration, such as healthy hone in osteosarcoma patients, after the delivery of drugs to kill cancer cells.
The rosette nanotubes may also be used in tissue engineering, where living cells are utilized as engineering materials. Applications for tissue engineering are used to repair or replace portions of whole tissues such as bone, cartilage, blood vessels, muscle, etc. Tissues are fabricated in the laboratory from combinations of engineered extracellular matrices (“scaffolds”), cells, and biologically active molecules destined for transplantation. For example, nasal chondrocytes can expand in culture to engineer a cartilage graft. The rosette nanotubes of the current disclosure can be used as scaffolds in tissue engineering methods, e.g. using nasal chondrocytes, as well as a transfer vehicle to deliver therapeutic agents to specific tissues, e.g. cartilage, when using tissue engineering techniques known to a skilled person in the art.
Genes and Proteins Used as Agents/Delivery Cargo
The following Genes and Proteins can be used as agents to complex with Nanotubes and Nanopieces:
The following Genes and Proteins can be used as target gene of siRNA which complex with Nanotubes and Nanopieces:
The mRNA transcript sequence encoding human ADAMTS-5, provided by Genbank Accession No. NM_007038.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 1).









1
ataaattcat tgttccacct cctcgcatct tcacagcgct cgcgctgctc tcggcgctcg



 


61
cagctgccga ctggggatga cggcgggcag gaggagaccg cagccgaagg gacacagaca


 


121
cgccgcttca ccagctcgcc tcaggctgcc cccctgcatt tttgttttaa tttttacggc


 


181
tttttcccct ctctttcttc ccttcctcct ggtcccagca gagccaagga aacccacaaa


 


241
ataagaaagg aagtgggccc cggagcttgg aacctccaca gccggcttgt ccagcgcagc


 


301
gcgggggcgg gaggctgcgc gcaccagttg ccagcccggt gcgcggtacc tttccttact


 


361
tttcttgaaa cagcgatcgt gcctgcattt ggtggttttt tggtttttgt ttttttcctt


 


421
ttcccgtatt tgctgaatct ccactatccg actttttttt tttaatcttt tctttccccc


 


481
cccccccacc ccacctcttt ctggagcacg aatccaaaca ttttcccaag caacaaagaa


 


541
aagttcgcac gctggcaccg cagcccggac aggctggcgc tgctgccggg cccccctccc


 


601
tccgacactt gactcaatcc tgcaagcaag tgtgtgtgtg tccccatccc ccgccccgtt


 


661
aacttcatag caaataacaa atacccataa agtcccagtc gcgcagcccc tccccgcggg


 


721
cagcgcacta tgctgctcgg gtgggcgtcc ctgctgctgt gcgcgttccg cctgcccctg


 


781
gccgcggtcg gccccgccgc gacacctgcc caggataaag ccgggcagcc tccgactgct


 


841
gcagcagccg cccagccccg ccggcggcag ggggaggagg tgcaggagcg agccgagcct


 


901
cccggccacc cgcaccccct ggcgcagcgg cgcaggagca aggggctggt gcagaacatc


 


961
gaccaactct actccggcgg cggcaaggtg ggctacctcg tctacgcggg cggccggagg


 


1021
ttcctcttgg acctggagcg agatggttcg gtgggcattg ctggcttcgt gcccgcagga


 


1081
ggcgggacga gtgcgccctg gcgccaccgg agccactgct tctatcgggg cacagtggac


 


1141
ggtagtcccc gctctctggc tgtctttgac ctctgtgggg gtctcgacgg cttcttcgcg


 


1201
gtcaagcacg cgcgctacac cctaaagcca ctgctgcgcg gaccctgggc ggaggaagaa


 


1261
aaggggcgcg tgtacgggga tgggtccgca cggatcctgc acgtctacac ccgcgagggc


 


1321
ttcagcttcg aggccctgcc gccgcgcgcc agctgcgaaa cccccgcgtc cacaccggag


 


1381
gcccacgagc atgctccggc gcacagcaac ccgagcggac gcgcagcact ggcctcgcag


 


1441
ctcttggacc agtccgctct ctcgcccgct gggggctcag gaccgcagac gtggtggcgg


 


1501
cggcggcgcc gctccatctc ccgggcccgc caggtggagc tgcttctggt ggctgacgcg


 


1561
tccatggcgc ggttgtatgg ccggggcctg cagcattacc tgctgaccct ggcctccatc


 


1621
gccaataggc tgtacagcca tgctagcatc gagaaccaca tccgcctggc cgtggtgaag


 


1681
gtggtggtgc taggcgacaa ggacaagagc ctggaagtga gcaagaacgc tgccaccaca


 


1741
ctcaagaact tttgcaagtg gcagcaccaa cacaaccagc tgggagatga ccatgaggag


 


1801
cactacgatg cagctatcct gtttactcgg gaggatttat gtgggcatca ttcatgtgac


 


1861
accctgggaa tggcagacgt tgggaccata tgttctccag agcgcagctg tgctgtgatt


 


1921
gaagacgatg gcctccacgc agccttcact gtggctcacg aaatcggaca tttacttggc


 


1981
ctctcccatg acgattccaa attctgtgaa gagacctttg gttccacaga agataagcgc


 


2041
ttaatgtctt ccatccttac cagcattgat gcatctaagc cctggtccaa atgcacttca


 


2101
gccaccatca cagaattcct ggatgatggc catggtaact gtttgctgga cctaccacga


 


2161
aagcagatcc tgggccccga agaactccca ggacagacct acgatgccac ccagcagtgc


 


2221
aacctgacat tcgggcctga gtactccgtg tgtcccggca tggatgtctg tgctcgcctg


 


2281
tggtgtgctg tggtacgcca gggccagatg gtctgtctga ccaagaagct gcctgcggtg


 


2341
gaagggacgc cttgtggaaa ggggagaatc tgcctgcagg gcaaatgtgt ggacaaaacc


 


2401
aagaaaaaat attattcaac gtcaagccat ggcaactggg gatcttgggg atcctggggc


 


2461
cagtgttctc gctcatgtgg aggaggagtg cagtttgcct atcgtcactg taataaccct


 


2521
gctcccagaa acaacggacg ctactgcaca gggaagaggg ccatctaccg ctcctgcagt


 


2581
ctcatgccct gcccacccaa tggtaaatca tttcgtcatg aacagtgtga ggccaaaaat


 


2641
ggctatcagt ctgatgcaaa aggagtcaaa acttttgtgg aatgggttcc caaatatgca


 


2701
ggtgtcctgc cagcggatgt gtgcaagctg acctgcagag ccaagggcac tggctactat


 


2761
gtggtatttt ctccaaaggt gaccgatggc actgaatgta ggctgtacag taattccgtc


 


2821
tgcgtccggg ggaagtgtgt gagaactggc tgtgacggca tcattggctc aaagctgcag


 


2881
tatgacaagt gcggagtatg tggaggagac aactccagct gtacaaagat tgttggaacc


 


2941
tttaataaga aaagtaaggg ttacactgac gtggtgagga ttcctgaagg ggcaacccac


 


3001
ataaaagttc gacagttcaa agccaaagac cagactagat tcactgccta tttagccctg


 


3061
aaaaagaaaa acggtgagta ccttatcaat ggaaagtaca tgatctccac ttcagagact


 


3121
atcattgaca tcaatggaac agtcatgaac tatagcggtt ggagccacag ggatgacttc


 


3181
ctgcatggca tgggctactc tgccacgaag gaaattctaa tagtgcagat tcttgcaaca


 


3241
gaccccacta aaccattaga tgtccgttat agcttttttg ttcccaagaa gtccactcca


 


3301
aaagtaaact ctgtcactag tcatggcagc aataaagtgg gatcacacac ttcgcagccg


 


3361
cagtgggtca cgggcccatg gctcgcctgc tctaggacct gtgacacagg ttggcacacc


 


3421
agaacggtgc agtgccagga tggaaaccgg aagttagcaa aaggatgtcc tctctcccaa


 


3481
aggccttctg cgtttaagca atgcttgttg aagaaatgtt agcctgtggt tatgatctta


 


3541
tgcacaaaga taactggagg attcagcact gatgcagtcg tggtgaacag gaggtctacc


 


3601
taacgcacag aaagtcatgc ttcagtgaca ttgtcaacag gagtccaatt atgggcagaa


 


3661
tctgctctct gtgaccaaaa gaggatgtgc actgcttcac gtgacagtgg tgaccttgca


 


3721
atatagaaaa acttgggagt tattgaacat cccctgggct tacaagaaac actgatgaat


 


3781
gtaaaatcag gggacatttg aagatggcag aactgtctcc cccttgtcac ctacctctga


 


3841
tagaatgtct ttaatggtat cataatcatt ttcacccata atacacagta gcttcttctt


 


3901
actgtttgta aatacattct cccttggtat gtcactttat atcccctggt tctattaaaa


 


3961
tatccatata tatttctata aaaaaagtgt ttgaccaaag taggtctgca gctatttcaa


 


4021
cttccttccg tttccagaaa gagctgtgga tattttactg gaaattaaga acttgctgct


 


4081
gttttaataa gatgtagtat attttctgac tacaggagat aaaatttcag tcaaaaaacc


 


4141
attttgacag caagtatctt ctgagaaatt ttgaaaagta aatagatctc agtgtatcta


 


4201
gtcacttaaa tacatacacg ggttcattta cttaaacctt tgactgcctg tatttttttc


 


4261
aggtagctag ccaaattaat gcataatttc agatgtagaa gtagggtttg cgtgtgtgtg


 


4321
tgtgatcata ctcaagagtc taaaaactag tttccttgtg ttggaaattt aaaaggaaaa


 


4381
aaatcgtatt tcactgtgtt ttcaatttat attttcacaa ctactttctc tctccagagc


 


4441
tttcatctga tatctcacaa tgtatgatat acgtacaaaa cacacagcaa gttttctatc


 


4501
atgtccaaca cattcaacac tggtatacct cctaccagca agcctttaaa atgcatttgt


 


4561
gtttgcttat ttgttttgtt caagggttca gtaagaccta caatgttttg tatttcttga


 


4621
cttattttat tagaaacatt aaagatcact tggtagttag ccacattgag aagtggttat


 


4681
cattgttaat gtggttaatg ccaaaaagtg gttaatatta ataagactgt ttccacacca


 


4741
taggcaataa tttcttaatt taaaaaatct aagtatattc ctattgtact aaatattttt


 


4801
cccaactgga aagcacttga ttgtacccgt aagtgtttga gtgatgacat gtgatgattt


 


4861
tcagaaagtt gttgtttttg tttccatagc ctgtttaagt aggttgtaag tttgaatagt


 


4921
tagacatgga aattatttta taagcacaca cctaaagata tctttttaga tgataaaatg


 


4981
tacacccccc catcaccaac ctcacaactt agaaaatcta agttgtttga tttctttggg


 


5041
atttcttttg ttgtgaaaca ctgcaaagcc aatttttctt tataaaaatt catagtaatc


 


5101
ctgccaaatg tgcctattgt taaagatttg catgtgaaga tcttagggaa ccactgtttg


 


5161
agttctacaa gctcatgaga gtttattttt attataagat gtttttaata taaaagaatt


 


5221
atgtaactga tcactatatt acatcatttc agtgggccag gaaaatagat gtcttgctgt


 


5281
tttcagtatt ttcttaagaa attgctttta aaacaaataa ttgttttaca aaaccaataa


 


5341
ttatcctttg aattttcata gactgacttt gcttttgacg tagaaatttt ttttctcaat


 


5401
aaattatcac tttgagaaat gaggcctgta caaggctgat aacctatatg tgatggagat


 


5461
cacccaatgc caagggcaga aagcaaacct agttaaatag gtgagaaaaa aaataataat


 


5521
cccagtgcca tttgtctgtg caaagagaat taggagagag gttaatgtta cttttttcca


 


5581
ttttggaaat aattttaatc aagtaactca aatgtgacaa aatttatttt tattttttgt


 


5641
ggttatattc ccaacaacat taaaaaatac tcgaggcata aatgtagttg tctcctactc


 


5701
tgcttctctt actatactca tacattttta atatggttta tcaatgattc atgtttccct


 


5761
caaatagtga tggtttacac ctgtcatgga aacaatccta gagagctcag agcaattaaa


 


5821
ccactattcc atgcttttaa gtagttttct ccaccttttt cttatgagtc tcactagatt


 


5881
gactgaggaa tgtatgtcta aattcctgga gaagatgata tggattggaa actgaaattc


 


5941
agagaaatgg agtgttcaat agataccacg aattgtgaac aaagggaaaa ttctatacaa


 


6001
ctcaatctaa gtcagtccac tttgacttcg tactgtcttt cacctttcca ttgttgcatc


 


6061
ttgaattttt taaaatgtct agaattcagg atgctagggg ctacttcttt aaaaaaaaaa


 


6121
aaaaaaaaga attcgtctga aaatgctcag gtttgtaaga atctaatctc acttacataa


 


6181
ctaagcactc cataataagt tttattaagt acaaagggag ccagaaaaaa tgacatttat


 


6241
ttcttctaga tcagaaaaat ttaaattaag ccctgccttg ctgtttagaa atatgtgggc


 


6301
attgttataa tttattcaat aaatttatgt tcctttgcct tcctgtggaa acagttttat


 


6361
cccactaaac taggaattag gggataaatc acaaacaaaa aaaaagttgc agcactgaaa


 


6421
aaaagtaatt tattgttttt gcaactggta tgtgaatttg tgtgataaaa ttatttattc


 


6481
ttatttaaca aaaatatgtt caaatttttc tatatttaaa atgttttgct gttgtcctac


 


6541
tttttaattt atgcttcatg tttgtgtata aagtacactt ttacactttg tgagtttaca


 


6601
taatatacag cactggttgc ttttgtattt ttttacagaa agctttctgt gtgaagcagg


 


6661
tgtatatgta tatattcctc atgtattctt attctgatac tatcattttt ctttccaagg


 


6721
aaattttaat ctgtcatgac caatagtgtt cattacttgt gcctatgata ataggttttt


 


6781
tacatcacat taacactatt ttttccaagt cacaaataag aaaaacactt attcaatgaa


 


6841
acaaggtgca agttttaaat ttgggtacac aaatagccta gaagcttcct acagacgcta


 


6901
agacacagcc aataatcaga tcctttcact tcatcgagaa acttggacaa gtcgatattg


 


6961
atgtattaga tgaaagttgt ctacacacaa cttctgaggg atacaaacga taataaaacc


 


7021
aaatgttgtc tgtttctcct ttagaaacac ctcctaaaat taatatcatt tagtctctag


 


7081
tgtctgtagg attctacaga tgagcacaaa tagattgggt ttgtataaca aatgctaata


 


7141
gtcataactg tttctacaaa tatggggtgt ccattaagag aatgtgatgt tttcctactg


 


7201
ctgttgaatc ccatggggtg attataggac ttgaaatagg cagagtcacc tctgatgaca


 


7261
tcagcttgcc tctgtgattt cacagtctga tcctggcaac aagacaaagc acccttggac


 


7321
acacagccaa tctctggttg tgatatttcc ccattgattc cttccttgtt aacaaggtca


 


7381
ttttaatggt tcaggtgagg acagcagcca gattcaaagt ccagaatttg tgctgttaca


 


7441
tagagttcac actgtcaaat aacattgaat ttaataatga tcaaattttt ctagtagtct


 


7501
ttggcagagt gtataatctc attggcatga ttggtgaata ttactaatct ctttataatg


 


7561
aaagatgctt tacaaatacc ttatatttgc taacatttca aaactactaa ataaatgaaa


 


7621
tagccatgtg tacagaaatg gtcatttaaa gctttaatag aaccaaattc aagacaatgt


 


7681
atcatttaga cacacagaaa aggaacttgt atgttttccc tattattttt ctcatttgcc


 


7741
aacaatctat agttttaggt tatcaaacag atagatcaac ttaactggct agtacattga


 


7801
aaaatcttcc taagaatcct ttgttagcat aatctataga gataatttct caaattatat


 


7861
catcatgatg catataaact ctataatgta taattgtgtt tcatttattt aatgtatgag


 


7921
aacatattga aatacaaaac catgcattag ccaaaaaatt ggaatacagg tagtgttcag


 


7981
atcagcaaaa cattcagtct ggtaaatgcc tgcctggggc tatgatatca ttctcaatgc


 


8041
aggttttatg gaaaaactaa aagaatatgt tgttagatga tgttggtttt gaaaaaaaaa


 


8101
agacattaac atacacatta gttagcccag ttaattgcat tctactaata tagttgcaca


 


8161
ttagcaataa ttttgctgtc tctggtcttt attttgtggc ttcaactaac tggaccatgt


 


8221
ggactgtaaa ggtcaaatgg aaaaaacgag cagtggcccc tcatcctgta aggtactgct


 


8281
acatcagagt gacctaaaag tctaacactg tgaggaaaac tgtgatttgt aggaaaaaaa


 


8341
aaaaaaacaa ataaaaaaca gggcatgctt tttaattttt ttccactttc ctttggcaca


 


8401
cccaatgaac aattctaatt tttattgagg tgctaacatc tttcgtgacc gactgtcaaa


 


8461
tgtggtattt ttgagttact atttttctac atgattttac agtttgcaag aaagacctct


 


8521
aagctttgtg tcacggtagg gcacaacttg atactcaaaa tttgaaaaat aagcacatcc


 


8581
aatgattgtt ttgaccaaca gtggtcagtg acgtaaactg catgtgcatc tgaggacatt


 


8641
taaggggtca ttaaaatttg aggagcatca ggccggagta gcagactttt agatgagtca


 


8701
tatttcagca ttcactaagt cctcagcatt ccattcaaac tgtcgtgtat atttggcctg


 


8761
attttttttc aagctttgca ataatttatg ttattggtaa acacttggtg actatatctc


 


8821
agccttttct ttaacaactc acaatatatt agaaacacgt ctacctatac tgagagtata


 


8881
tttacaatag aagaacatac tgtatgtgac tttgtaaagc tagacttttg attaagaaat


 


8941
atataatctc tggatgctat ttttgcatta tacactcagg cacaacgtaa accttgatgg


 


9001
ctcatcttgc tacaattacg agttgaaaaa cactacttac gtatttgtat gacctattag


 


9061
tcagaggaaa tcatacatat gctttgtaaa tagactttgc agataactaa atagactgaa


 


9121
gaaatatgtt gcatttgata gaagcaattg cataaatatt tggtttctat attagagtct


 


9181
gtgagtaaag tcaagtaata aacctaagta ggtataacag atttttaaac cttgaaactt


 


9241
gctttgatgg tagagaaaat cattgaagat ttacatactg tatataagat gtaaaatgta


 


9301
cgctgcttat taccctcaat tttccagaag caatggtata taatgcagtt gaaaaaccaa


 


9361
aaatcttgga aaactaagac gggtcttgtt taaaatgtct ctcagctttg gcaaccttca


 


9421
aatcttaatc aactatttaa agcattactg tgtcttgtag cctgcattcc acaacagctc


 


9481
tgttattcag gtaaaagact tgaactgagc cgtttgggac ctatactgta atattttcat


 


9541
tgaggaacaa tatcctattt tgtaaagcat ttccctatgt gtgactttaa actgtaaaat


 


9601
taaacactgc ttttgtgggt tcagtgggca taataaatat aaattgtaaa ctaggttaaa


 


9661
gta






The amino acid sequence of human ADAMTS-5 (preproprotein), provided by Genbank Accession No. NP_008969.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 2).









1
mllgwaslll cafrlplaav gpaatpaqdk agqpptaaaa aqprrrqgee vqeraeppgh



 


61
phplaqrrrs kglvqnidql ysgggkvgyl vyaggrrfll dlerdgsvgi agfvpagggt


 


121
sapwrhrshc fyrgtvdgsp rslavfdlcg gldgffavkh arytlkpllr gpwaeeekgr


 


181
vygdgsaril hvytregfsf ealpprasce tpastpeahe hapahsnpsg raalasqlld


 


241
qsalspaggs gpqtwwrrrr rsisrarqve lllvadasma rlygrglqhy lltlasianr


 


301
lyshasienh irlavvkvvv lgdkdkslev sknaattlkn fckwqhqhnq lgddheehyd


 


361
aailftredl cghhscdtlg madvgticsp erscaviedd glhaaftvah eighllglsh


 


421
ddskfceetf gstedkrlms siltsidask pwskctsati teflddghgn clldlprkqi


 


481
lgpeelpgqt ydatqqcnlt fgpeysvcpg mdvcarlwca vvrqgqmvcl tkklpavegt


 


541
pcgkgriclq gkcvdktkkk yystsshgnw gswgswgqcs rscgggvqfa yrhcnnpapr


 


601
nngryctgkr aiyrscslmp cppngksfrh eqceakngyq sdakgvktfv ewvpkyagvl


 


661
padvckltcr akgtgyyvvf spkvtdgtec rlysnsvcvr gkcvrtgcdg iigsklqydk


 


721
cgvcggdnss ctkivgtfnk kskgytdvvr ipegathikv rqfkakdqtr ftaylalkkk


 


781
ngeylingky mistsetiid ingtvmnysg wshrddflhg mgysatkeil ivqilatdpt


 


841
kpldvrysff vpkkstpkvn svtshgsnkv gshtsqpqwv tgpwlacsrt cdtgwhtrtv


 


901
qcqdgnrkla kgcplsqrps afkqcllkkc


 








(Signal peptide AA 1-6; proprotein AA 17-930; mature peptide AA 262-930).







The siRNA used to target human ADAMTS-5 mRNA include following sequences (SEQ ID NO: 3-6):









SEQ NO: 3:



5′-GCUCAAAGCUGCAGUAUGA-3′


 



SEQ NO: 4:



5′-GAAGUCCACUCCAAAAGUA-3′


 



SEQ NO: 5:



5′-GCACUACGAUGCAGCUAUC-3′


 



SEQ NO: 6:



5′-CGAAGGAAAUUCUAAUAGU-3′






The molecular beacon used to target human ADAMTS-5 mRNA includes the following sequences (SEQ ID NO: 7-9):







SEQ NO 7:


5′-CCGGTC TAACATTTCTTCAACAAGCA GACCGG-3′


 


SEQ NO 8:


5′-CCGGTC TTATACACAAACATGAAGCA GACCGG-3′


 


SEQ NO 9:


5′-CCGGTC TACATCTTATTAAAACAGCA GACCGG-3′






The mRNA transcript sequence encoding human ADAMTS-4, provided by Genbank Accession No. NM_005099.4, is incorporated herein by reference, and is shown below (SEQ ID NO: 10).









1
ggggagaacc cacagggaga cccacagaca catatgcacg agagagacag aggaggaaag



 


61
agacagagac aaaggcacag cggaagaagg cagagacagg gcaggcacag aagcggccca


 


121
gacagagtcc tacagaggga gaggccagag aagctgcaga agacacaggc agggagagac


 


181
aaagatccag gaaaggaggg ctcaggagga gagtttggag aagccagacc cctgggcacc


 


241
tctcccaagc ccaaggacta agttttctcc atttccttta acggtcctca gcccttctga


 


301
aaactttgcc tctgaccttg gcaggagtcc aagcccccag gctacagaga ggagctttcc


 


361
aaagctaggg tgtggaggac ttggtgccct agacggcctc agtccctccc agctgcagta


 


421
ccagtgccat gtcccagaca ggctcgcatc ccgggagggg cttggcaggg cgctggctgt


 


481
ggggagccca accctgcctc ctgctcccca ttgtgccgct ctcctggctg gtgtggctgc


 


541
ttctgctact gctggcctct ctcctgccct cagcccggct ggccagcccc ctcccccggg


 


601
aggaggagat cgtgtttcca gagaagctca acggcagcgt cctgcctggc tcgggcgccc


 


661
ctgccaggct gttgtgccgc ttgcaggcct ttggggagac gctgctacta gagctggagc


 


721
aggactccgg tgtgcaggtc gaggggctga cagtgcagta cctgggccag gcgcctgagc


 


781
tgctgggtgg agcagagcct ggcacctacc tgactggcac catcaatgga gatccggagt


 


841
cggtggcatc tctgcactgg gatgggggag ccctgttagg cgtgttacaa tatcgggggg


 


901
ctgaactcca cctccagccc ctggagggag gcacccctaa ctctgctggg ggacctgggg


 


961
ctcacatcct acgccggaag agtcctgcca gcggtcaagg tcccatgtgc aacgtcaagg


 


1021
ctcctcttgg aagccccagc cccagacccc gaagagccaa gcgctttgct tcactgagta


 


1081
gatttgtgga gacactggtg gtggcagatg acaagatggc cgcattccac ggtgcggggc


 


1141
taaagcgcta cctgctaaca gtgatggcag cagcagccaa ggccttcaag cacccaagca


 


1201
tccgcaatcc tgtcagcttg gtggtgactc ggctagtgat cctggggtca ggcgaggagg


 


1261
ggccccaagt ggggcccagt gctgcccaga ccctgcgcag cttctgtgcc tggcagcggg


 


1321
gcctcaacac ccctgaggac tcggaccctg accactttga cacagccatt ctgtttaccc


 


1381
gtcaggacct gtgtggagtc tccacttgcg acacgctggg tatggctgat gtgggcaccg


 


1441
tctgtgaccc ggctcggagc tgtgccattg tggaggatga tgggctccag tcagccttca


 


1501
ctgctgctca tgaactgggt catgtcttca acatgctcca tgacaactcc aagccatgca


 


1561
tcagtttgaa tgggcctttg agcacctctc gccatgtcat ggcccctgtg atggctcatg


 


1621
tggatcctga ggagccctgg tccccctgca gtgcccgctt catcactgac ttcctggaca


 


1681
atggctatgg gcactgtctc ttagacaaac cagaggctcc attgcatctg cctgtgactt


 


1741
tccctggcaa ggactatgat gctgaccgcc agtgccagct gaccttcggg cccgactcac


 


1801
gccattgtcc acagctgccg ccgccctgtg ctgccctctg gtgctctggc cacctcaatg


 


1861
gccatgccat gtgccagacc aaacactcgc cctgggccga tggcacaccc tgcgggcccg


 


1921
cacaggcctg catgggtggt cgctgcctcc acatggacca gctccaggac ttcaatattc


 


1981
cacaggctgg tggctggggt ccttggggac catggggtga ctgctctcgg acctgtgggg


 


2041
gtggtgtcca gttctcctcc cgagactgca cgaggcctgt cccccggaat ggtggcaagt


 


2101
actgtgaggg ccgccgtacc cgcttccgct cctgcaacac tgaggactgc ccaactggct


 


2161
cagccctgac cttccgcgag gagcagtgtg ctgcctacaa ccaccgcacc gacctcttca


 


2221
agagcttccc agggcccatg gactgggttc ctcgctacac aggcgtggcc ccccaggacc


 


2281
agtgcaaact cacctgccag gcccaggcac tgggctacta ctatgtgctg gagccacggg


 


2341
tggtagatgg gaccccctgt tccccggaca gctcctcggt ctgtgtccag ggccgatgca


 


2401
tccatgctgg ctgtgatcgc atcattggct ccaagaagaa gtttgacaag tgcatggtgt


 


2461
gcggagggga cggttctggt tgcagcaagc agtcaggctc cttcaggaaa ttcaggtacg


 


2521
gatacaacaa tgtggtcact atccccgcgg gggccaccca cattcttgtc cggcagcagg


 


2581
gaaaccctgg ccaccggagc atctacttgg ccctgaagct gccagatggc tcctatgccc


 


2641
tcaatggtga atacacgctg atgccctccc ccacagatgt ggtactgcct ggggcagtca


 


2701
gcttgcgcta cagcggggcc actgcagcct cagagacact gtcaggccat gggccactgg


 


2761
cccagccttt gacactgcaa gtcctagtgg ctggcaaccc ccaggacaca cgcctccgat


 


2821
acagcttctt cgtgccccgg ccgacccctt caacgccacg ccccactccc caggactggc


 


2881
tgcaccgaag agcacagatt ctggagatcc ttcggcggcg cccctgggcg ggcaggaaat


 


2941
aacctcacta tcccggctgc cctttctggg caccggggcc tcggacttag ctgggagaaa


 


3001
gagagagctt ctgttgctgc ctcatgctaa gactcagtgg ggaggggctg tgggcgtgag


 


3061
acctgcccct cctctctgcc ctaatgcgca ggctggccct gccctggttt cctgccctgg


 


3121
gaggcagtga tgggttagtg gatggaaggg gctgacagac agccctccat ctaaactgcc


 


3181
ccctctgccc tgcgggtcac aggagggagg gggaaggcag ggagggcctg ggccccagtt


 


3241
gtatttattt agtatttatt cacttttatt tagcaccagg gaaggggaca aggactaggg


 


3301
tcctggggaa cctgacccct gacccctcat agccctcacc ctggggctag gaaatccagg


 


3361
gtggtggtga taggtataag tggtgtgtgt atgcgtgtgt gtgtgtgtga aaatgtgtgt


 


3421
gtgcttatgt atgaggtaca acctgttctg ctttcctctt cctgaatttt attttttggg


 


3481
aaaagaaaag tcaagggtag ggtgggcctt cagggagtga gggattatct tttttttttt


 


3541
ttctttcttt ctttcttttt tttttttgag acagaatctc gctctgtcgc ccaggctgga


 


3601
gtgcaatggc acaatctcgg ctcactgcat cctccgcctc ccgggttcaa gtgattctca


 


3661
tgcctcagcc tcctgagtag ctgggattac aggctcctgc caccacgccc ggctaatttt


 


3721
tgttttgttt tgtttggaga cagagtctcg ctattgtcac cagggctgga atgatttcag


 


3781
ctcactgcaa ccttcgccac ctgggttcca gcaattctcc tgcctcagcc tcccgagtag


 


3841
ctgagattat aggcacctac caccacgccc ggctaatttt tgtattttta gtagagacgg


 


3901
ggtttcacca tgttggccag gctggtctcg aactcctgac cttaggtgat ccactcgcct


 


3961
tcatctccca aagtgctggg attacaggcg tgagccaccg tgcctggcca cgcccaacta


 


4021
atttttgtat ttttagtaga gacagggttt caccatgttg gccaggctgc tcttgaactc


 


4081
ctgacctcag gtaatcgacc tgcctcggcc tcccaaagtg ctgggattac aggtgtgagc


 


4141
caccacgccc ggtacatatt ttttaaattg aattctacta tttatgtgat ccttttggag


 


4201
tcagacagat gtggttgcat cctaactcca tgtctctgag cattagattt ctcatttgcc


 


4261
aataataata cctcccttag aagtttgttg tgaggattaa ataatgtaaa taaagaacta


 


4321
gcataacact caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa


 


4381
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa






The amino acid sequence of human ADAMTS-4 (preproprotein), provided by Genbank Accession No. NP_005090.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 11).









1
msqtgshpgr glagrwlwga qpclllpivp lswlvwllll llasllpsar lasplpreee



 


61
ivfpeklngs vlpgsgapar llcrlqafge tllleleqds gvqvegltvq ylgqapellg


 


121
gaepgtyltg tingdpesva slhwdggall gvlqyrgael hlqpleggtp nsaggpgahi


 


181
lrrkspasgq gpmcnvkapl gspsprprra krfaslsrfv etlvvaddkm aafhgaglkr


 


241
ylltvmaaaa kafkhpsirn pvslvvtrlv ilgsgeegpq vgpsaaqtlr sfcawqrgln


 


301
tpedsdpdhf dtailftrqd lcgvstcdtl gmadvgtvcd parscaived dglqsaftaa


 


361
helghvfnml hdnskpcisl ngplstsrhv mapvmahvdp eepwspcsar fitdfldngy


 


421
ghclldkpea plhlpvtfpg kdydadrqcq ltfgpdsrhc pqlpppcaal wcsghlngha


 


481
mcqtkhspwa dgtpcgpaqa cmggrclhmd qlqdfnipqa ggwgpwgpwg dcsrtcgggv


 


541
qfssrdctrp vprnggkyce grrtrfrscn tedcptgsal tfreeqcaay nhrtdlfksf


 


601
pgpmdwvpry tgvapqdqck ltcqaqalgy yyvleprvvd gtpcspdsss vcvqgrciha


 


661
gcdriigskk kfdkcmvcgg dgsgcskqsg sfrkfrygyn nvvtipagat hilvrqqgnp


 


721
ghrsiylalk lpdgsyalng eytlmpsptd vvlpgayslr ysgataaset lsghgplaqp


 


781
ltlqvlvagn pqdtrlrysf fvprptpstp rptpqdwlhr raqileilrr rpwagrk






The siRNA used to target human ADAMTS-4 mRNA includes the following sequences (SEQ ID NO: 12-15):









SEQ NO: 12:



5′-CCGCAAUCCUGUCAGCUUG-3′


 



SEQ NO: 13:



5′-GCGCUUUGCUUCACUGAGU-3′


 



SEQ NO: 14:



5′-GGACACACGCCUCCGAUAC-3′


 



SEQ NO: 15:



5′-GCACCGAAGAGCACAGAUU-3′






The molecular beacon used to target human ADAMTS-4 mRNA includes the following sequences (SEQ ID NO: 16-18):









SEQ NO: 16:



5′-CCGGTC TTTTCACACACACACACACGGACCGG-3′


 



SEQ NO: 17:



5′-CCGGTC TAAAAATACAAAAATTAGCCGACCGG-3′


 



SEQ NO: 18:



5′-CCGGTC TTGTCTCTGTCTCTTTCCTCGACCGG-3′






The mRNA transcript sequence encoding human MMP-13, provided by Genbank Accession No. NM_002427.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 19).









1
acaacagtcc ccaggcatca ccattcaaga tgcatccagg ggtcctggct gccttcctct



 


61
tcttgagctg gactcattgt cgggccctgc cccttcccag tggtggtgat gaagatgatt


 


121
tgtctgagga agacctccag tttgcagagc gctacctgag atcatactac catcctacaa


 


181
atctcgcggg aatcctgaag gagaatgcag caagctccat gactgagagg ctccgagaaa


 


241
tgcagtcttt cttcggctta gaggtgactg gcaaacttga cgataacacc ttagatgtca


 


301
tgaaaaagcc aagatgcggg gttcctgatg tgggtgaata caatgttttc cctcgaactc


 


361
ttaaatggtc caaaatgaat ttaacctaca gaattgtgaa ttacacccct gatatgactc


 


421
attctgaagt cgaaaaggca ttcaaaaaag ccttcaaagt ttggtccgat gtaactcctc


 


481
tgaattttac cagacttcac gatggcattg ctgacatcat gatctctttt ggaattaagg


 


541
agcatggcga cttctaccca tttgatgggc cctctggcct gctggctcat gcttttcctc


 


601
ctgggccaaa ttatggagga gatgcccatt ttgatgatga tgaaacctgg acaagtagtt


 


661
ccaaaggcta caacttgttt cttgttgctg cgcatgagtt cggccactcc ttaggtcttg


 


721
accactccaa ggaccctgga gcactcatgt ttcctatcta cacctacacc ggcaaaagcc


 


781
actttatgct tcctgatgac gatgtacaag ggatccagtc tctctatggt ccaggagatg


 


841
aagaccccaa ccctaaacat ccaaaaacgc cagacaaatg tgacccttcc ttatcccttg


 


901
atgccattac cagtctccga ggagaaacaa tgatctttaa agacagattc ttctggcgcc


 


961
tgcatcctca gcaggttgat gcggagctgt ttttaacgaa atcattttgg ccagaacttc


 


1021
ccaaccgtat tgatgctgca tatgagcacc cttctcatga cctcatcttc atcttcagag


 


1081
gtagaaaatt ttgggctctt aatggttatg acattctgga aggttatccc aaaaaaatat


 


1141
ctgaactggg tcttccaaaa gaagttaaga agataagtgc agctgttcac tttgaggata


 


1201
caggcaagac tctcctgttc tcaggaaacc aggtctggag atatgatgat actaaccata


 


1261
ttatggataa agactatccg agactaatag aagaagactt cccaggaatt ggtgataaag


 


1321
tagatgctgt ctatgagaaa aatggttata tctatttttt caacggaccc atacagtttg


 


1381
aatacagcat ctggagtaac cgtattgttc gcgtcatgcc agcaaattcc attttgtggt


 


1441
gttaagtgtc tttttaaaaa ttgttattta aatcctgaag agcatttggg gtaatacttc


 


1501
cagaagtgcg gggtagggga agaagagcta tcaggagaaa gcttggttct gtgaacaagc


 


1561
ttcagtaagt tatctttgaa tatgtagtat ctatatgact atgcgtggct ggaaccacat


 


1621
tgaagaatgt tagagtaatg aaatggagga tctctaaaga gcatctgatt cttgttgctg


 


1681
tacaaaagca atggttgatg atacttccca caccacaaat gggacacatg gtctgtcaat


 


1741
gagagcataa tttaaaaata tatttataag gaaattttac aagggcataa agtaaataca


 


1801
tgcatataat gaataaatca ttcttactaa aaagtataaa atagtatgaa aatggaaatt


 


1861
tgggagagcc atacataaaa gaaataaacc aaaggaaaat gtctgtaata atagactgta


 


1921
acttccaaat aaataatttt cattttgcac tgaggatatt cagatgtatg tgcccttctt


 


1981
cacacagaca ctaacgaaat atcaaagtca ttaaagacag gagacaaaag agcagtggta


 


2041
agaatagtag atgtggcctt tgaattctgt ttaattttca cttttggcaa tgactcaaag


 


2101
tctgctctca tataagacaa atattccttt gcatattata aaggataaag aaggatgatg


 


2161
tctttttatt aaaatatttc aggttcttca gaagtcacac attacaaagt taaaattgtt


 


2221
atcaaaatag tctaaggcca tggcatccct ttttcataaa ttatttgatt atttaagact


 


2281
aaaagttgca ttttaaccct attttaccta gctaattatt taattgtcca gtttgtcttg


 


2341
gatatatagg ctattttcta aagacttgta tagcatgaaa taaaatatat cttataaagt


 


2401
ggaagtatgt atattaaaaa agagacatcc aaattttttt ttaaagcagt ctactagatt


 


2461
gtgatccctt gagatatgga aggatgcctt tttttctctg catttaaaaa aatcccccag


 


2521
cacttcccac agtgcctatt gatacttggg gagggtgctt ggcacttatt gaatatatga


 


2581
tcggccatca agggaagaac tattgtgctc agagacactg ttgataaaaa ctcaggcaaa


 


2641
gaaaatgaaa tgcatatttg caaagtgtat taggaagtgt ttatgttgtt tataataaaa


 


2701
atatattttc aacagacaaa aaaaaaaaaa aaaaa






The amino acid sequence of human MMP-13 (collagenase 3 preproprotein), provided by Genbank Accession No. NP_002418.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 20).









1
mhpgvlaafl flswthcral plpsggdedd lseedlqfae rylrsyyhpt nlagilkena



 


61
assmterlre mqsffglevt gklddntldv mkkprcgvpd vgeynvfprt lkwskmnlty


 


121
rivnytpdmt hsevekafkk afkvwsdvtp lnftrlhdgi adimisfgik ehgdfypfdg


 


181
psgllahafp pgpnyggdah fdddetwtss skgynlflva ahefghslgl dhskdpgalm


 


241
fpiytytgks hfmlpdddvq giqslygpgd edpnpkhpkt pdkcdpslsl daitslrget


 


301
mifkdrffwr lhpqqvdael fltksfwpel pnridaayeh pshdlififr grkfwalngy


 


361
dilegypkki selglpkevk kisaavhfed tgktllfsgn qvwryddtnh imdkdyprli


 


421
eedfpgigdk vdavyekngy iyffngpiqf eysiwsnriv rvmpansilw c


 








(Signal protein AA 1-19; proprotein AA 20-471; mature peptide AA 104-471).







The siRNA used to target human MMP-13 mRNA includes the following sequences (SEQ ID NO: 21-24):









SEQ NO: 21:



5′-UUUCACACACACACACACGC-3′


 



SEQ NO: 22:



5′-UUUUCACACACACACACACG-3′


 



SEQ NO: 23:



5′-UAAAAAUACAAAAAUUAGCC-3′


 



SEQ NO: 24:



5′-UUUGUCUCUGUCUCUUUCCU-3′






The molecular beacon used to target human MMP-13 mRNA includes the following sequences (SEQ ID NO: 25-27):









SEQ NO 25:



5′-CCGGTC TACACACACCACTTATACCT GACCGG-3′


 



SEQ NO 26:



5′-CCGGTC TATAATCTCAGCTACTCGGG GACCGG-3′


 



SEQ NO 27:



5′-CCGGTC AAACAAAACAAAAATTAGCC GACCGG-3′






The mRNA transcript sequence encoding human MMP-1 variant 2, provided by Genbank Accession No. NM_001145938.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 28).









1
agcatgagtc agacagcctc tggctttctg gaagggcaag gactctatat atacagaggg



 


61
agcttcctag ctgggatatt ggagcagcaa gaggctggga agccatcact taccttgcac


 


121
tgagaaagaa gacaaaggca agttgaaaag cggagaaata gtggcccagt ggttgaaaaa


 


181
ttgaagcaaa tgcaggaatt ctttgggctg aaagtgactg ggaaaccaga tgctgaaacc


 


241
ctgaaggtga tgaagcagcc cagatgtgga gtgcctgatg tggctcagtt tgtcctcact


 


301
gaggggaacc ctcgctggga gcaaacacat ctgacctaca ggattgaaaa ttacacgcca


 


361
gatttgccaa gagcagatgt ggaccatgcc attgagaaag ccttccaact ctggagtaat


 


421
gtcacacctc tgacattcac caaggtctct gagggtcaag cagacatcat gatatctttt


 


481
gtcaggggag atcatcggga caactctcct tttgatggac ctggaggaaa tcttgctcat


 


541
gcttttcaac caggcccagg tattggaggg gatgctcatt ttgatgaaga tgaaaggtgg


 


601
accaacaatt tcagagagta caacttacat cgtgttgcag ctcatgaact cggccattct


 


661
cttggactct cccattctac tgatatcggg gctttgatgt accctagcta caccttcagt


 


721
ggtgatgttc agctagctca ggatgacatt gatggcatcc aagccatata tggacgttcc


 


781
caaaatcctg tccagcccat cggcccacaa accccaaaag cgtgtgacag taagctaacc


 


841
tttgatgcta taactacgat tcggggagaa gtgatgttct ttaaagacag attctacatg


 


901
cgcacaaatc ccttctaccc ggaagttgag ctcaatttca tttctgtttt ctggccacaa


 


961
ctgccaaatg ggcttgaagc tgcttacgaa tttgccgaca gagatgaagt ccggtttttc


 


1021
aaagggaata agtactgggc tgttcaggga cagaatgtgc tacacggata ccccaaggac


 


1081
atctacagct cctttggctt ccctagaact gtgaagcata tcgatgctgc tctttctgag


 


1141
gaaaacactg gaaaaaccta cttctttgtt gctaacaaat actggaggta tgatgaatat


 


1201
aaacgatcta tggatccagg ttatcccaaa atgatagcac atgactttcc tggaattggc


 


1261
cacaaagttg atgcagtttt catgaaagat ggatttttct atttctttca tggaacaaga


 


1321
caatacaaat ttgatcctaa aacgaagaga attttgactc tccagaaagc taatagctgg


 


1381
ttcaactgca ggaaaaattg aacattacta atttgaatgg aaaacacatg gtgtgagtcc


 


1441
aaagaaggtg ttttcctgaa gaactgtcta ttttctcagt catttttaac ctctagagtc


 


1501
actgatacac agaatataat cttatttata cctcagtttg catatttttt tactatttag


 


1561
aatgtagccc tttttgtact gatataattt agttccacaa atggtgggta caaaaagtca


 


1621
agtttgtggc ttatggattc atataggcca gagttgcaaa gatcttttcc agagtatgca


 


1681
actctgacgt tgatcccaga gagcagcttc agtgacaaac atatcctttc aagacagaaa


 


1741
gagacaggag acatgagtct ttgccggagg aaaagcagct caagaacaca tgtgcagtca


 


1801
ctggtgtcac cctggatagg caagggataa ctcttctaac acaaaataag tgttttatgt


 


1861
ttggaataaa gtcaaccttg tttctactgt tttatacact ttc






The amino acid sequence of human MMP-1 (interstitial collagenase isoform 2), provided by Genbank Accession No. NP_001139410.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 29).









1
mqeffglkvt gkpdaetlkv mkqprcgvpd vaqfvltegn prweqthlty rienytpdlp



 


61
radvdhaiek afqlwsnvtp ltftkvsegq adimisfvrg dhrdnspfdg pggnlahafq


 


121
pgpgiggdah fdederwtnn freynlhrva ahelghslgl shstdigalm ypsytfsgdv


 


181
qlaqddidgi qaiygrsqnp vqpigpqtpk acdskltfda ittirgevmf fkdrfymrtn


 


241
pfypevelnf isvfwpqlpn gleaayefad rdevrffkgn kywavqgqnv lhgypkdiys


 


301
sfgfprtvkh idaalseent gktyffvank ywrydeykrs mdpgypkmia hdfpgighkv


 


361
davfmkdgff yffhgtrqyk fdpktkrilt lqkanswfnc rkn






The siRNA used to target human MMP-1 variant 1 mRNA include following sequences (SEQ ID NO: 30-33):









SEQ NO: 30:



5′-UUAGCUUACUGUCACACGC-3′


 



SEQ NO: 31:



5′-UUAUAUUCAUCAUACCUCC-3′


 



SEQ NO: 32:



5′-UUGUCUUCUUUCUCAGUGC-3′


 



SEQ NO: 33:



5′-UUCGUAAGCAGCUUCAAGC-3′






The molecular beacon used to target human MMP-1 variant 1 mRNA includes the following sequences (SEQ ID NO: 34-36):









SEQ NO 34:



5′-CCGGTC TTCGTAAGCAGCTTCAAGC GACCGG-3′


 



SEQ NO 35:



5′-CCGGTC TAAAGAACATCACTTTCC GACCGG-3′


 



SEQ NO 36:



5′-CCGGTC TAAAACAGTAGAAACAAGG GACCGG-3′






The mRNA transcript sequence encoding human MMP-9, provided by Genbank Accession No. NM_004994.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 37).









1
agacacctct gccctcacca tgagcctctg gcagcccctg gtcctggtgc tcctggtgct



 


61
gggctgctgc tttgctgccc ccagacagcg ccagtccacc cttgtgctct tccctggaga


 


121
cctgagaacc aatctcaccg acaggcagct ggcagaggaa tacctgtacc gctatggtta


 


181
cactcgggtg gcagagatgc gtggagagtc gaaatctctg gggcctgcgc tgctgcttct


 


241
ccagaagcaa ctgtccctgc ccgagaccgg tgagctggat agcgccacgc tgaaggccat


 


301
gcgaacccca cggtgcgggg tcccagacct gggcagattc caaacctttg agggcgacct


 


361
caagtggcac caccacaaca tcacctattg gatccaaaac tactcggaag acttgccgcg


 


421
ggcggtgatt gacgacgcct ttgcccgcgc cttcgcactg tggagcgcgg tgacgccgct


 


481
caccttcact cgcgtgtaca gccgggacgc agacatcgtc atccagtttg gtgtcgcgga


 


541
gcacggagac gggtatccct tcgacgggaa ggacgggctc ctggcacacg cctttcctcc


 


601
tggccccggc attcagggag acgcccattt cgacgatgac gagttgtggt ccctgggcaa


 


661
gggcgtcgtg gttccaactc ggtttggaaa cgcagatggc gcggcctgcc acttcccctt


 


721
catcttcgag ggccgctcct actctgcctg caccaccgac ggtcgctccg acggcttgcc


 


781
ctggtgcagt accacggcca actacgacac cgacgaccgg tttggcttct gccccagcga


 


841
gagactctac acccaggacg gcaatgctga tgggaaaccc tgccagtttc cattcatctt


 


901
ccaaggccaa tcctactccg cctgcaccac ggacggtcgc tccgacggct accgctggtg


 


961
cgccaccacc gccaactacg accgggacaa gctcttcggc ttctgcccga cccgagctga


 


1021
ctcgacggtg atggggggca actcggcggg ggagctgtgc gtcttcccct tcactttcct


 


1081
gggtaaggag tactcgacct gtaccagcga gggccgcgga gatgggcgcc tctggtgcgc


 


1141
taccacctcg aactttgaca gcgacaagaa gtggggcttc tgcccggacc aaggatacag


 


1201
tttgttcctc gtggcggcgc atgagttcgg ccacgcgctg ggcttagatc attcctcagt


 


1261
gccggaggcg ctcatgtacc ctatgtaccg cttcactgag gggcccccct tgcataagga


 


1321
cgacgtgaat ggcatccggc acctctatgg tcctcgccct gaacctgagc cacggcctcc


 


1381
aaccaccacc acaccgcagc ccacggctcc cccgacggtc tgccccaccg gaccccccac


 


1441
tgtccacccc tcagagcgcc ccacagctgg ccccacaggt cccccctcag ctggccccac


 


1501
aggtcccccc actgctggcc cttctacggc cactactgtg cctttgagtc cggtggacga


 


1561
tgcctgcaac gtgaacatct tcgacgccat cgcggagatt gggaaccagc tgtatttgtt


 


1621
caaggatggg aagtactggc gattctctga gggcaggggg agccggccgc agggcccctt


 


1681
ccttatcgcc gacaagtggc ccgcgctgcc ccgcaagctg gactcggtct ttgaggagcg


 


1741
gctctccaag aagcttttct tcttctctgg gcgccaggtg tgggtgtaca caggcgcgtc


 


1801
ggtgctgggc ccgaggcgtc tggacaagct gggcctggga gccgacgtgg cccaggtgac


 


1861
cggggccctc cggagtggca gggggaagat gctgctgttc agcgggcggc gcctctggag


 


1921
gttcgacgtg aaggcgcaga tggtggatcc ccggagcgcc agcgaggtgg accggatgtt


 


1981
ccccggggtg cctttggaca cgcacgacgt cttccagtac cgagagaaag cctatttctg


 


2041
ccaggaccgc ttctactggc gcgtgagttc ccggagtgag ttgaaccagg tggaccaagt


 


2101
gggctacgtg acctatgaca tcctgcagtg ccctgaggac tagggctccc gtcctgcttt


 


2161
ggcagtgcca tgtaaatccc cactgggacc aaccctgggg aaggagccag tttgccggat


 


2221
acaaactggt attctgttct ggaggaaagg gaggagtgga ggtgggctgg gccctctctt


 


2281
ctcacctttg ttttttgttg gagtgtttct aataaacttg gattctctaa cctttaaaaa


 


2341
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa






The amino acid sequence of human MMP-9 (preproprotein), provided by Genbank Accession No. NP_004985.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 38).









1
mslwqplvlv llvlgccfaa prqrqstlvl fpgdlrtnlt drqlaeeyly rygytrvaem



 


61
rgeskslgpa llllqkqlsl petgeldsat lkamrtprcg vpdlgrfqtf egdlkwhhhn


 


121
itywiqnyse dlpravidda farafalwsa vtpltftrvy srdadiviqf gvaehgdgyp


 


181
fdgkdgllah afppgpgiqg dahfdddelw slgkgvvvpt rfgnadgaac hfpfifegrs


 


241
ysacttdgrs dglpwcstta nydtddrfgf cpserlytqd gnadgkpcqf pfifqgqsys


 


301
acttdgrsdg yrwcattany drdklfgfcp tradstvmgg nsagelcvfp ftflgkeyst


 


361
ctsegrgdgr lwcattsnfd sdkkwgfcpd qgyslflvaa hefghalgld hssvpealmy


 


421
pmyrftegpp lhkddvngir hlygprpepe prppttttpq ptapptvcpt gpptvhpser


 


481
ptagptgpps agptgpptag pstattvpls pvddacnvni fdaiaeignq lylfkdgkyw


 


541
rfsegrgsrp qgpfliadkw palprkldsv feerlskklf ffsgrqvwvy tgasvlgprr


 


601
ldklglgadv aqvtgalrsg rgkmllfsgr rlwrfdvkaq mvdprsasev drmfpgvpld


 


661
thdvfqyrek ayfcqdrfyw rvssrselnq vdqvgyvtyd ilqcped


 








(signal protein AA 1-19; proportein AA 20-707; mature protein 107-707)







The siRNA used to target human MMP-9 mRNA include following sequences (SEQ ID NO: 39-42):









SEQ NO: 39:



5′-UUGUCGCUGUCAAAGUUCGAG-3′


 



SEQ NO: 40:



5′-UUCUUGUCGCUGUCAAAGUUC-3′


 



SEQ NO: 41:



5′-UUCAACUCACUCCGGGAACUC-3′


 



SEQ NO: 42:



5′-UUCACGUCGUCCUUAUGCAAG-3′






The molecular beacon used to target human MMP-9 mRNA includes the following sequences (SEQ ID NO:43-45):











SEQ NO: 43:




5′-CCGGTC TTGTCGCTGTCAAAGTTCGGACCGG-3′



 




SEQ NO: 44:




5′-CCGGTC TTATTAGAAACACTCCAAC GACCGG-3′



 




SEQ NO: 45:




5′-CCGGTC ATTCACGTCGTCCTTATGC GACCGG-3′






The mRNA transcript sequence encoding human MMP-3, provided by Genbank Accession No. NM_002422.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 46).









1
ctacaaggag gcaggcaaga cagcaaggca tagagacaac atagagctaa gtaaagccag



 


61
tggaaatgaa gagtcttcca atcctactgt tgctgtgcgt ggcagtttgc tcagcctatc


 


121
cattggatgg agctgcaagg ggtgaggaca ccagcatgaa ccttgttcag aaatatctag


 


181
aaaactacta cgacctcaaa aaagatgtga aacagtttgt taggagaaag gacagtggtc


 


241
ctgttgttaa aaaaatccga gaaatgcaga agttccttgg attggaggtg acggggaagc


 


301
tggactccga cactctggag gtgatgcgca agcccaggtg tggagttcct gatgttggtc


 


361
acttcagaac ctttcctggc atcccgaagt ggaggaaaac ccaccttaca tacaggattg


 


421
tgaattatac accagatttg ccaaaagatg ctgttgattc tgctgttgag aaagctctga


 


481
aagtctggga agaggtgact ccactcacat tctccaggct gtatgaagga gaggctgata


 


541
taatgatctc ttttgcagtt agagaacatg gagactttta cccttttgat ggacctggaa


 


601
atgttttggc ccatgcctat gcccctgggc cagggattaa tggagatgcc cactttgatg


 


661
atgatgaaca atggacaaag gatacaacag ggaccaattt atttctcgtt gctgctcatg


 


721
aaattggcca ctccctgggt ctctttcact cagccaacac tgaagctttg atgtacccac


 


781
tctatcactc actcacagac ctgactcggt tccgcctgtc tcaagatgat ataaatggca


 


841
ttcagtccct ctatggacct ccccctgact cccctgagac ccccctggta cccacggaac


 


901
ctgtccctcc agaacctggg acgccagcca actgtgatcc tgctttgtcc tttgatgctg


 


961
tcagcactct gaggggagaa atcctgatct ttaaagacag gcacttttgg cgcaaatccc


 


1021
tcaggaagct tgaacctgaa ttgcatttga tctcttcatt ttggccatct cttccttcag


 


1081
gcgtggatgc cgcatatgaa gttactagca aggacctcgt tttcattttt aaaggaaatc


 


1141
aattctgggc tatcagagga aatgaggtac gagctggata cccaagaggc atccacaccc


 


1201
taggtttccc tccaaccgtg aggaaaatcg atgcagccat ttctgataag gaaaagaaca


 


1261
aaacatattt ctttgtagag gacaaatact ggagatttga tgagaagaga aattccatgg


 


1321
agccaggctt tcccaagcaa atagctgaag actttccagg gattgactca aagattgatg


 


1381
ctgtttttga agaatttggg ttcttttatt tctttactgg atcttcacag ttggagtttg


 


1441
acccaaatgc aaagaaagtg acacacactt tgaagagtaa cagctggctt aattgttgaa


 


1501
agagatatgt agaaggcaca atatgggcac tttaaatgaa gctaataatt cttcacctaa


 


1561
gtctctgtga attgaaatgt tcgttttctc ctgcctgtgc tgtgactcga gtcacactca


 


1621
agggaacttg agcgtgaatc tgtatcttgc cggtcatttt tatgttatta cagggcattc


 


1681
aaatgggctg ctgcttagct tgcaccttgt cacatagagt gatctttccc aagagaaggg


 


1741
gaagcactcg tgtgcaacag acaagtgact gtatctgtgt agactatttg cttatttaat


 


1801
aaagacgatt tgtcagttat tttatctt






The amino acid sequence of human MMP-3 (preproprotein), provided by Genbank Accession No. NP_002413.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 47).









1
mkslpillll cvavcsaypl dgaargedts mnlvqkylen yydlkkdvkq fvrrkdsgpv



 


61
vkkiremqkf lglevtgkld sdtlevmrkp rcgvpdvghf rtfpgipkwr kthltyrivn


 


121
ytpdlpkdav dsavekalkv weevtpltfs rlyegeadim isfavrehgd fypfdgpgnv


 


181
lahayapgpg ingdahfddd eqwtkdttgt nlflvaahei ghslglfhsa ntealmyply


 


241
hsltdltrfr lsqddingiq slygpppdsp etplvptepv ppepgtpanc dpalsfdavs


 


301
tlrgeilifk drhfwrkslr klepelhlis sfwpslpsgv daayevtskd lvfifkgnqf


 


361
wairgnevra gyprgihtlg fpptvrkida aisdkeknkt yffvedkywr fdekrnsmep


 


421
gfpkqiaedf pgidskidav feefgffyff tgssqlefdp nakkvthtlk snswlnc


 








(signal peptide AA 1-17; proprotein AA 18-477; mature protein AA 100-477).







The siRNA used to target human MMP-3 mRNA include following sequences (SEQ ID NO: 48-51):









SEQ NO: 48:



5′-UUCAUCAUCAUCAAAGUGGG-3′


 



SEQ NO: 49:



5′-UAAUAACAUAAAAAUGACCG-3′


 



SEQ NO: 50:



5′-UAGUCUACACAGAUACAGUC-3′


 



SEQ NO: 51:



5′-UAUAUCAUCUUGAGACAGGC-3′






The molecular beacon used to target human MMP-3 mRNA includes the following sequences (SEQ ID NO: 52-54):









SEQ NO 52:



5′-CCGGTC TATATCATCTTGAGACAGGC GACCGG-3′


 



SEQ NO 53:



5′-CCGGTC TTTCTCTTCTCATCAAATCT GACCGG-3′


 



SEQ NO 54:



5′-CCGGTC TAACAAACTGTTTCACATCT GACCGG-3′






The mRNA transcript sequence encoding human IL-1 alpha, provided by Genbank Accession No. NM_000575.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 55).









1
accaggcaac accattgaag gctcatatgt aaaaatccat gccttccttt ctcccaatct



 


61
ccattcccaa acttagccac tggcttctgg ctgaggcctt acgcatacct cccggggctt


 


121
gcacacacct tcttctacag aagacacacc ttgggcatat cctacagaag accaggcttc


 


181
tctctggtcc ttggtagagg gctactttac tgtaacaggg ccagggtgga gagttctctc


 


241
ctgaagctcc atcccctcta taggaaatgt gttgacaata ttcagaagag taagaggatc


 


301
aagacttctt tgtgctcaaa taccactgtt ctcttctcta ccctgcccta accaggagct


 


361
tgtcacccca aactctgagg tgatttatgc cttaatcaag caaacttccc tcttcagaaa


 


421
agatggctca ttttccctca aaagttgcca ggagctgcca agtattctgc caattcaccc


 


481
tggagcacaa tcaacaaatt cagccagaac acaactacag ctactattag aactattatt


 


541
attaataaat tcctctccaa atctagcccc ttgacttcgg atttcacgat ttctcccttc


 


601
ctcctagaaa cttgataagt ttcccgcgct tccctttttc taagactaca tgtttgtcat


 


661
cttataaagc aaaggggtga ataaatgaac caaatcaata acttctggaa tatctgcaaa


 


721
caacaataat atcagctatg ccatctttca ctattttagc cagtatcgag ttgaatgaac


 


781
atagaaaaat acaaaactga attcttccct gtaaattccc cgttttgacg acgcacttgt


 


841
agccacgtag ccacgcctac ttaagacaat tacaaaaggc gaagaagact gactcaggct


 


901
taagctgcca gccagagagg gagtcatttc attggcgttt gagtcagcaa agaagtcaag


 


961
atggccaaag ttccagacat gtttgaagac ctgaagaact gttacagtga aaatgaagaa


 


1021
gacagttcct ccattgatca tctgtctctg aatcagaaat ccttctatca tgtaagctat


 


1081
ggcccactcc atgaaggctg catggatcaa tctgtgtctc tgagtatctc tgaaacctct


 


1141
aaaacatcca agcttacctt caaggagagc atggtggtag tagcaaccaa cgggaaggtt


 


1201
ctgaagaaga gacggttgag tttaagccaa tccatcactg atgatgacct ggaggccatc


 


1261
gccaatgact cagaggaaga aatcatcaag cctaggtcag caccttttag cttcctgagc


 


1321
aatgtgaaat acaactttat gaggatcatc aaatacgaat tcatcctgaa tgacgccctc


 


1381
aatcaaagta taattcgagc caatgatcag tacctcacgg ctgctgcatt acataatctg


 


1441
gatgaagcag tgaaatttga catgggtgct tataagtcat caaaggatga tgctaaaatt


 


1501
accgtgattc taagaatctc aaaaactcaa ttgtatgtga ctgcccaaga tgaagaccaa


 


1561
ccagtgctgc tgaaggagat gcctgagata cccaaaacca tcacaggtag tgagaccaac


 


1621
ctcctcttct tctgggaaac tcacggcact aagaactatt tcacatcagt tgcccatcca


 


1681
aacttgttta ttgccacaaa gcaagactac tgggtgtgct tggcaggggg gccaccctct


 


1741
atcactgact ttcagatact ggaaaaccag gcgtaggtct ggagtctcac ttgtctcact


 


1801
tgtgcagtgt tgacagttca tatgtaccat gtacatgaag aagctaaatc ctttactgtt


 


1861
agtcatttgc tgagcatgta ctgagccttg taattctaaa tgaatgttta cactctttgt


 


1921
aagagtggaa ccaacactaa catataatgt tgttatttaa agaacaccct atattttgca


 


1981
tagtaccaat cattttaatt attattcttc ataacaattt taggaggacc agagctactg


 


2041
actatggcta ccaaaaagac tctacccata ttacagatgg gcaaattaag gcataagaaa


 


2101
actaagaaat atgcacaata gcagttgaaa caagaagcca cagacctagg atttcatgat


 


2161
ttcatttcaa ctgtttgcct tctactttta agttgctgat gaactcttaa tcaaatagca


 


2221
taagtttctg ggacctcagt tttatcattt tcaaaatgga gggaataata cctaagcctt


 


2281
cctgccgcaa cagtttttta tgctaatcag ggaggtcatt ttggtaaaat acttcttgaa


 


2341
gccgagcctc aagatgaagg caaagcacga aatgttattt tttaattatt atttatatat


 


2401
gtatttataa atatatttaa gataattata atatactata tttatgggaa ccccttcatc


 


2461
ctctgagtgt gaccaggcat cctccacaat agcagacagt gttttctggg ataagtaagt


 


2521
ttgatttcat taatacaggg cattttggtc caagttgtgc ttatcccata gccaggaaac


 


2581
tctgcattct agtacttggg agacctgtaa tcatataata aatgtacatt aattaccttg


 


2641
agccagtaat tggtccgatc tttgactctt ttgccattaa acttacctgg gcattcttgt


 


2701
ttcaattcca cctgcaatca agtcctacaa gctaaaatta gatgaactca actttgacaa


 


2761
ccatgagacc actgttatca aaactttctt ttctggaatg taatcaatgt ttcttctagg


 


2821
ttctaaaaat tgtgatcaga ccataatgtt acattattat caacaatagt gattgataga


 


2881
gtgttatcag tcataactaa ataaagcttg caacaaaatt ctctgacaaa aaaaaaaaaa


 


2941
aaa






The amino acid sequence of human IL-1 alpha (proprotein), provided by Genbank Accession No. NP_000566.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 56).









1
makvpdmfed lkncysenee dsssidhlsl nqksfyhvsy gplhegcmdq syslsisets



 


61
ktskltfkes mvvvatngkv lkkrrlslsq sitdddleai andseeeiik prsapfsfls


 


121
nvkynfmrii kyefilndal nqsiirandq yltaaalhnl deavkfdmga yksskddaki


 


181
tvilrisktq lyvtaqdedq pvllkempei pktitgsetn llffwethgt knyftsvahp


 


241
nlfiatkqdy wvclaggpps itdfqilenq a


 








(mature peptide AA 113-271).







The siRNA used to target human IL-1 alpha mRNA include following sequences (SEQ ID NO: 57-60):









SEQ NO: 57:



5′-UUUCUAUGUUCAUUCAACUC-3′



 



SEQ NO: 58:



5′-UCAUUCAACUCGAUACUGGC-3′



 



SEQ NO: 59:



5′-UUCAUUCAACUCGAUACUGG-3′



 



SEQ NO: 60:



5′-UAAUAGUUCUAAUAGUAGCU-3′






The molecular beacon used to target human IL-1 alpha mRNA includes the following sequences (SEQ ID NO: 61-63):









SEQ NO 61:



5′-CCGGTC TTTCTTAGTTTTCTTATGCC GACCGG-3′



 



SEQ NO 62:



5′-CCGGTC TAATAGTTCTAATAGTAGC GACCGG-3′



 



SEQ NO 63:



5′-CCGGTC TATGAACTGTCAACACTGC GACCGG-3′






The mRNA transcript sequence encoding human IL-1 beta, provided by Genbank Accession No. NM_000576.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 64).









1
accaaacctc ttcgaggcac aaggcacaac aggctgctct gggattctct tcagccaatc



 


61
ttcattgctc aagtgtctga agcagccatg gcagaagtac ctgagctcgc cagtgaaatg


 


121
atggcttatt acagtggcaa tgaggatgac ttgttctttg aagctgatgg ccctaaacag


 


181
atgaagtgct ccttccagga cctggacctc tgccctctgg atggcggcat ccagctacga


 


241
atctccgacc accactacag caagggcttc aggcaggccg cgtcagttgt tgtggccatg


 


301
gacaagctga ggaagatgct ggttccctgc ccacagacct tccaggagaa tgacctgagc


 


361
accttctttc ccttcatctt tgaagaagaa cctatcttct tcgacacatg ggataacgag


 


421
gcttatgtgc acgatgcacc tgtacgatca ctgaactgca cgctccggga ctcacagcaa


 


481
aaaagcttgg tgatgtctgg tccatatgaa ctgaaagctc tccacctcca gggacaggat


 


541
atggagcaac aagtggtgtt ctccatgtcc tttgtacaag gagaagaaag taatgacaaa


 


601
atacctgtgg ccttgggcct caaggaaaag aatctgtacc tgtcctgcgt gttgaaagat


 


661
gataagccca ctctacagct ggagagtgta gatcccaaaa attacccaaa gaagaagatg


 


721
gaaaagcgat ttgtcttcaa caagatagaa atcaataaca agctggaatt tgagtctgcc


 


781
cagttcccca actggtacat cagcacctct caagcagaaa acatgcccgt cttcctggga


 


841
gggaccaaag gcggccagga tataactgac ttcaccatgc aatttgtgtc ttcctaaaga


 


901
gagctgtacc cagagagtcc tgtgctgaat gtggactcaa tccctagggc tggcagaaag


 


961
ggaacagaaa ggtttttgag tacggctata gcctggactt tcctgttgtc tacaccaatg


 


1021
cccaactgcc tgccttaggg tagtgctaag aggatctcct gtccatcagc caggacagtc


 


1081
agctctctcc tttcagggcc aatccccagc ccttttgttg agccaggcct ctctcacctc


 


1141
tcctactcac ttaaagcccg cctgacagaa accacggcca catttggttc taagaaaccc


 


1201
tctgtcattc gctcccacat tctgatgagc aaccgcttcc ctatttattt atttatttgt


 


1261
ttgtttgttt tattcattgg tctaatttat tcaaaggggg caagaagtag cagtgtctgt


 


1321
aaaagagcct agtttttaat agctatggaa tcaattcaat ttggactggt gtgctctctt


 


1381
taaatcaagt cctttaatta agactgaaaa tatataagct cagattattt aaatgggaat


 


1441
atttataaat gagcaaatat catactgttc aatggttctg aaataaactt cactgaag






The amino acid sequence of human IL-1 beta (proprotein), provided by Genbank Accession No. NP_000567.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 65).









1
maevpelase mmayysgned dlffeadgpk qmkcsfqdld lcpldggiql risdhhyskg



 


61
frqaasvvva mdklrkmlvp cpqtfqendl stffpfifee epiffdtwdn eayvhdapvr


 


121
slnctlrdsq qkslvmsgpy elkalhlqgq dmeqqvvfsm sfvqgeesnd kipvalglke


 


181
knlylscvlk ddkptlqles vdpknypkkk mekrfvfnki einnklefes aqfpnwyist


 


241
sqaenmpvfl ggtkggqdit dftmqfvss


 








(mature peptide AA 117-269)







The siRNA used to target human IL-1 beta mRNA includes the following sequences (SEQ ID NO: 66-69):









SEQ NO: 66:



5′-UUAUCAUCUUUCAACACGCAG-3′



 



SEQ NO: 67:



5′-UUUUACAGACACUGCUACUUC-3′



 



SEQ NO: 68:



5′-UUUGUCAUUACUUUCUUCUCC-3′



 



SEQ NO: 69:



5′-UACAGACACUGCUACUUCUUG-3′






The molecular beacon used to target human IL-1 beta mRNA includes the following sequences (SEQ ID NO: 70-72):









SEQ NO: 70:



5′-CCGGTC TTTTGTCATTACTTTCTTCTC GACCGG-3′



 



SEQ NO: 71:



5′-CCGGTC TTTCAGTCTTAATTAAAGGAC GACCGG-3′



 



SEQ NO: 72:



5′-CCGGTC TTACATAAATTAACTCAGCT GACCGG-3′






The mRNA transcript sequence encoding human IL-6, provided by Genbank Accession No. NM_00600.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 73).









1
aatattagag tctcaacccc caataaatat aggactggag atgtctgagg ctcattctgc



 


61
cctcgagccc accgggaacg aaagagaagc tctatctccc ctccaggagc ccagctatga


 


121
actccttctc cacaagcgcc ttcggtccag ttgccttctc cctggggctg ctcctggtgt


 


181
tgcctgctgc cttccctgcc ccagtacccc caggagaaga ttccaaagat gtagccgccc


 


241
cacacagaca gccactcacc tcttcagaac gaattgacaa acaaattcgg tacatcctcg


 


301
acggcatctc agccctgaga aaggagacat gtaacaagag taacatgtgt gaaagcagca


 


361
aagaggcact ggcagaaaac aacctgaacc ttccaaagat ggctgaaaaa gatggatgct


 


421
tccaatctgg attcaatgag gagacttgcc tggtgaaaat catcactggt cttttggagt


 


481
ttgaggtata cctagagtac ctccagaaca gatttgagag tagtgaggaa caagccagag


 


541
ctgtgcagat gagtacaaaa gtcctgatcc agttcctgca gaaaaaggca aagaatctag


 


601
atgcaataac cacccctgac ccaaccacaa atgccagcct gctgacgaag ctgcaggcac


 


661
agaaccagtg gctgcaggac atgacaactc atctcattct gcgcagcttt aaggagttcc


 


721
tgcagtccag cctgagggct cttcggcaaa tgtagcatgg gcacctcaga ttgttgttgt


 


781
taatgggcat tccttcttct ggtcagaaac ctgtccactg ggcacagaac ttatgttgtt


 


841
ctctatggag aactaaaagt atgagcgtta ggacactatt ttaattattt ttaatttatt


 


901
aatatttaaa tatgtgaagc tgagttaatt tatgtaagtc atatttatat ttttaagaag


 


961
taccacttga aacattttat gtattagttt tgaaataata atggaaagtg gctatgcagt


 


1021
ttgaatatcc tttgtttcag agccagatca tttcttggaa agtgtaggct tacctcaaat


 


1081
aaatggctaa cttatacata tttttaaaga aatatttata ttgtatttat ataatgtata


 


1141
aatggttttt ataccaataa atggcatttt aaaaaattca gcaaaaaaaa aaaaaaaaaa


 


1201
a






The amino acid sequence of human IL-6 (precursor), provided by Genbank Accession No. NP_000591.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 74).









1
mnsfstsafg pvafslglll vlpaafpapv ppgedskdva aphrqpltss eridkqiryi



 


61
ldgisalrke tcnksnmces skealaennl nlpkmaekdg cfqsgfneet clvkiitgll


 


121
efevyleylq nrfesseeqa ravqmstkvl iqflqkkakn ldaittpdpt tnaslltklq


 


181
aqnqwlqdmt thlilrsfke flqsslralr qm


 








(Signal peptide AA 1-29; mature peptide AA 30-212).







The siRNA used to target human IL-6 mRNA include following sequences (SEQ ID NO: 75-78):









SEQ NO: 75:



5′-UAAAAUAGUGUCCUAACGCUC-3′



 



SEQ NO: 76:



5′-UCACUACUCUCAAAUCUGUUC-3′



 



SEQ NO: 77:



5′-UUACUCUUGUUACAUGUCUCC-3′



 



SEQ NO: 78:



5′-UAACGCUCAUACUUUUAGUUC-3′






The molecular beacon used to target human IL-6 mRNA includes the following sequences (SEQ ID NO: 79-81):









SEQ NO 79:



5′-CCGGTC TTACTCTTGTTACATGTCYCC GACCTT-3′



 



SEQ NO 80:



5′-CCGGTC TTACTCTTGTTACATGTCTCC GACCTT-3′



 



SEQ NO 81:



5′-CCGGTC TACATAAAATGTTTCAAGTGG GACCTT-3′






The mRNA transcript sequence encoding human IL-8, provided by Genbank Accession No. NM_000584.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 82).









1
gagggtgcat aagttctcta gtagggtgat gatataaaaa gccaccggag cactccataa



 


61
ggcacaaact ttcagagaca gcagagcaca caagcttcta ggacaagagc caggaagaaa


 


121
ccaccggaag gaaccatctc actgtgtgta aacatgactt ccaagctggc cgtggctctc


 


181
ttggcagcct tcctgatttc tgcagctctg tgtgaaggtg cagttttgcc aaggagtgct


 


241
aaagaactta gatgtcagtg cataaagaca tactccaaac ctttccaccc caaatttatc


 


301
aaagaactga gagtgattga gagtggacca cactgcgcca acacagaaat tattgtaaag


 


361
ctttctgatg gaagagagct ctgtctggac cccaaggaaa actgggtgca gagggttgtg


 


421
gagaagtttt tgaagagggc tgagaattca taaaaaaatt cattctctgt ggtatccaag


 


481
aatcagtgaa gatgccagtg aaacttcaag caaatctact tcaacacttc atgtattgtg


 


541
tgggtctgtt gtagggttgc cagatgcaat acaagattcc tggttaaatt tgaatttcag


 


601
taaacaatga atagtttttc attgtaccat gaaatatcca gaacatactt atatgtaaag


 


661
tattatttat ttgaatctac aaaaaacaac aaataatttt taaatataag gattttccta


 


721
gatattgcac gggagaatat acaaatagca aaattgaggc caagggccaa gagaatatcc


 


781
gaactttaat ttcaggaatt gaatgggttt gctagaatgt gatatttgaa gcatcacata


 


841
aaaatgatgg gacaataaat tttgccataa agtcaaattt agctggaaat cctggatttt


 


901
tttctgttaa atctggcaac cctagtctgc tagccaggat ccacaagtcc ttgttccact


 


961
gtgccttggt ttctccttta tttctaagtg gaaaaagtat tagccaccat cttacctcac


 


1021
agtgatgttg tgaggacatg tggaagcact ttaagttttt tcatcataac ataaattatt


 


1081
ttcaagtgta acttattaac ctatttatta tttatgtatt tatttaagca tcaaatattt


 


1141
gtgcaagaat ttggaaaaat agaagatgaa tcattgattg aatagttata aagatgttat


 


1201
agtaaattta ttttatttta gatattaaat gatgttttat tagataaatt tcaatcaggg


 


1261
tttttagatt aaacaaacaa acaattgggt acccagttaa attttcattt cagataaaca


 


1321
acaaataatt ttttagtata agtacattat tgtttatctg aaattttaat tgaactaaca


 


1381
atcctagttt gatactccca gtcttgtcat tgccagctgt gttggtagtg ctgtgttgaa


 


1441
ttacggaata atgagttaga actattaaaa cagccaaaac tccacagtca atattagtaa


 


1501
tttcttgctg gttgaaactt gtttattatg tacaaataga ttcttataat attatttaaa


 


1561
tgactgcatt tttaaataca aggctttata tttttaactt taagatgttt ttatgtgctc


 


1621
tccaaatttt ttttactgtt tctgattgta tggaaatata aaagtaaata tgaaacattt


 


1681
aaaatataat ttgttgtcaa agtaaaaaaa aaaaaaaa






The amino acid sequence of human IL-8(precursor), provided by Genbank Accession No. NP_000575.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 83).









1
mtsklavall aaflisaalc egavlprsak elrcqcikty skpfhpkfik elrviesgph



 


61
canteiivkl sdgrelcldp kenwvqrvve kflkraens






The siRNA used to target human IL-8 mRNA include following sequences (SEQ ID NO: 84-87):









SEQ NO: 84:



5′-UUUGUUUAAUCUAAAAACCC-3′



 



SEQ NO: 85:



5′-UUUACACACAGUGAGAUGGU-3′



 



SEQ NO: 86:



5′-UUCAAAUAUCACAUUCUAGC-3′



 



SEQ NO: 87:



5′-UUAUGCACUGACAUCUAAGU-3′






The molecular beacon used to target human IL-8 mRNA includes the following sequences (SEQ ID NO: 88-90):









SEQ NO 88:



5′-CCGGTC TATCACATTCTAGCAAACCC GACCGG-3′



 



SEQ NO 89:



5′-CCGGTC TACTAGAGAACTTATGCACC GACCGG-3′



 



SEQ NO 90:



5′-CCGGTC TAGTTCTAACTCATTATTCC GACCGG-3′






The mRNA transcript sequence encoding human IL-1R type 1 variant 1, provided by Genbank Accession No. NM_000877.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 91).









1
gtggccggcg gccggagccg actcggagcg cgcggcgccg gccgggagga gccggagagc



 


61
ggccgggccg ggcggtgggg gcgccggcct gccccgcgcg ccccagggag cggcaggaat


 


121
gtgacaatcg cgcgcccgcg caccgaagca ctcctcgctc ggctcctagg gctctcgccc


 


181
ctctgagctg agccgggttc cgcccggggc tgggatccca tcaccctcca cggccgtccg


 


241
tccaggtaga cgcaccctct gaagatggtg actccctcct gagaagctgg accccttggt


 


301
aaaagacaag gccttctcca agaagaatat gaaagtgtta ctcagactta tttgtttcat


 


361
agctctactg atttcttctc tggaggctga taaatgcaag gaacgtgaag aaaaaataat


 


421
tttagtgtca tctgcaaatg aaattgatgt tcgtccctgt cctcttaacc caaatgaaca


 


481
caaaggcact ataacttggt ataaagatga cagcaagaca cctgtatcta cagaacaagc


 


541
ctccaggatt catcaacaca aagagaaact ttggtttgtt cctgctaagg tggaggattc


 


601
aggacattac tattgcgtgg taagaaattc atcttactgc ctcagaatta aaataagtgc


 


661
aaaatttgtg gagaatgagc ctaacttatg ttataatgca caagccatat ttaagcagaa


 


721
actacccgtt gcaggagacg gaggacttgt gtgcccttat atggagtttt ttaaaaatga


 


781
aaataatgag ttacctaaat tacagtggta taaggattgc aaacctctac ttcttgacaa


 


841
tatacacttt agtggagtca aagataggct catcgtgatg aatgtggctg aaaagcatag


 


901
agggaactat acttgtcatg catcctacac atacttgggc aagcaatatc ctattacccg


 


961
ggtaatagaa tttattactc tagaggaaaa caaacccaca aggcctgtga ttgtgagccc


 


1021
agctaatgag acaatggaag tagacttggg atcccagata caattgatct gtaatgtcac


 


1081
cggccagttg agtgacattg cttactggaa gtggaatggg tcagtaattg atgaagatga


 


1141
cccagtgcta ggggaagact attacagtgt ggaaaatcct gcaaacaaaa gaaggagtac


 


1201
cctcatcaca gtgcttaata tatcggaaat tgaaagtaga ttttataaac atccatttac


 


1261
ctgttttgcc aagaatacac atggtataga tgcagcatat atccagttaa tatatccagt


 


1321
cactaatttc cagaagcaca tgattggtat atgtgtcacg ttgacagtca taattgtgtg


 


1381
ttctgttttc atctataaaa tcttcaagat tgacattgtg ctttggtaca gggattcctg


 


1441
ctatgatttt ctcccaataa aagcttcaga tggaaagacc tatgacgcat atatactgta


 


1501
tccaaagact gttggggaag ggtctacctc tgactgtgat atttttgtgt ttaaagtctt


 


1561
gcctgaggtc ttggaaaaac agtgtggata taagctgttc atttatggaa gggatgacta


 


1621
cgttggggaa gacattgttg aggtcattaa tgaaaacgta aagaaaagca gaagactgat


 


1681
tatcatttta gtcagagaaa catcaggctt cagctggctg ggtggttcat ctgaagagca


 


1741
aatagccatg tataatgctc ttgttcagga tggaattaaa gttgtcctgc ttgagctgga


 


1801
gaaaatccaa gactatgaga aaatgccaga atcgattaaa ttcattaagc agaaacatgg


 


1861
ggctatccgc tggtcagggg actttacaca gggaccacag tctgcaaaga caaggttctg


 


1921
gaagaatgtc aggtaccaca tgccagtcca gcgacggtca ccttcatcta aacaccagtt


 


1981
actgtcacca gccactaagg agaaactgca aagagaggct cacgtgcctc tcgggtagca


 


2041
tggagaagtt gccaagagtt ctttaggtgc ctcctgtctt atggcgttgc aggccaggtt


 


2101
atgcctcatg ctgacttgca gagttcatgg aatgtaacta tatcatcctt tatccctgag


 


2161
gtcacctgga atcagattat taagggaata agccatgacg tcaatagcag cccagggcac


 


2221
ttcagagtag agggcttggg aagatctttt aaaaaggcag taggcccggt gtggtggctc


 


2281
acgcctataa tcccagcact ttgggaggct gaagtgggtg gatcaccaga ggtcaggagt


 


2341
tcgagaccag cccagccaac atggcaaaac cccatctcta ctaaaaatac aaaaatgagc


 


2401
taggcatggt ggcacacgcc tgtaatccca gctacacctg aggctgaggc aggagaattg


 


2461
cttgaaccgg ggagacggag gttgcagtga gccgagtttg ggccactgca ctctagcctg


 


2521
gcaacagagc aagactccgt ctcaaaaaaa gggcaataaa tgccctctct gaatgtttga


 


2581
actgccaaga aaaggcatgg agacagcgaa ctagaagaaa gggcaagaag gaaatagcca


 


2641
ccgtctacag atggcttagt taagtcatcc acagcccaag ggcggggcta tgccttgtct


 


2701
ggggaccctg tagagtcact gaccctggag cggctctcct gagaggtgct gcaggcaaag


 


2761
tgagactgac acctcactga ggaagggaga catattcttg gagaactttc catctgcttg


 


2821
tattttccat acacatcccc agccagaagt tagtgtccga agaccgaatt ttattttaca


 


2881
gagcttgaaa actcacttca atgaacaaag ggattctcca ggattccaaa gttttgaagt


 


2941
catcttagct ttccacagga gggagagaac ttaaaaaagc aacagtagca gggaattgat


 


3001
ccacttctta atgctttcct ccctggcatg accatcctgt cctttgttat tatcctgcat


 


3061
tttacgtctt tggaggaaca gctccctagt ggcttcctcc gtctgcaatg tcccttgcac


 


3121
agcccacaca tgaaccatcc ttcccatgat gccgctcttc tgtcatcccg ctcctgctga


 


3181
aacacctccc aggggctcca cctgttcagg agctgaagcc catgctttcc caccagcatg


 


3241
tcactcccag accacctccc tgccctgtcc tccagcttcc cctcgctgtc ctgctgtgtg


 


3301
aattcccagg ttggcctggt ggccatgtcg cctgccccca gcactcctct gtctctgctc


 


3361
ttgcctgcac ccttcctcct cctttgccta ggaggccttc tcgcattttc tctagctgat


 


3421
cagaatttta ccaaaattca gaacatcctc caattccaca gtctctggga gactttccct


 


3481
aagaggcgac ttcctctcca gccttctctc tctggtcagg cccactgcag agatggtggt


 


3541
gagcacatct gggaggctgg tctccctcca gctggaattg ctgctctctg agggagaggc


 


3601
tgtggtggct gtctctgtcc ctcactgcct tccaggagca atttgcacat gtaacataga


 


3661
tttatgtaat gctttatgtt taaaaacatt ccccaattat cttatttaat ttttgcaatt


 


3721
attctaattt tatatataga gaaagtgacc tattttttaa aaaaatcaca ctctaagttc


 


3781
tattgaacct aggacttgag cctccatttc tggcttctag tctggtgttc tgagtacttg


 


3841
atttcaggtc aataacggtc ccccctcact ccacactggc acgtttgtga gaagaaatga


 


3901
cattttgcta ggaagtgacc gagtctagga atgcttttat tcaagacacc aaattccaaa


 


3961
cttctaaatg ttggaatttt caaaaattgt gtttagattt tatgaaaaac tcttctactt


 


4021
tcatctattc tttccctaga ggcaaacatt tcttaaaatg tttcattttc attaaaaatg


 


4081
aaagccaaat ttatatgcca ccgattgcag gacacaagca cagttttaag agttgtatga


 


4141
acatggagag gacttttggt ttttatattt ctcgtattta atatgggtga acaccaactt


 


4201
ttatttggaa taataatttt cctcctaaac aaaaacacat tgagtttaag tctctgactc


 


4261
ttgcctttcc acctgctttc tcctgggccc gctttgcctg cttgaaggaa cagtgctgtt


 


4321
ctggagctgc tgttccaaca gacagggcct agctttcatt tgacacacag actacagcca


 


4381
gaagcccatg gagcagggat gtcacgtctt gaaaagccta ttagatgttt tacaaattta


 


4441
attttgcaga ttattttagt ctgtcatcca gaaaatgtgt cagcatgcat agtgctaaga


 


4501
aagcaagcca atttggaaac ttaggttagt gacaaaattg gccagagagt gggggtgatg


 


4561
atgaccaaga attacaagta gaatggcagc tggaatttaa ggagggacaa gaatcaatgg


 


4621
ataagcgtgg gtggaggaag atccaaacag aaaagtgcaa agttattccc catcttccaa


 


4681
gggttgaatt ctggaggaag aagacacatt cctagttccc cgtgaacttc ctttgactta


 


4741
ttgtccccac taaaacaaaa caaaaaactt ttaatgcctt ccacattaat tagattttct


 


4801
tgcagttttt ttatggcatt tttttaaaga tgccctaagt gttgaagaag agtttgcaaa


 


4861
tgcaacaaaa tatttaatta ccggttgtta aaactggttt agcacaattt atattttccc


 


4921
tctcttgcct ttcttatttg caataaaagg tattgagcca ttttttaaat gacatttttg


 


4981
ataaattatg tttgtactag ttgatgaagg agtttttttt aacctgttta tataattttg


 


5041
cagcagaagc caaatttttt gtatattaaa gcaccaaatt catgtacagc atgcatcacg


 


5101
gatcaataga ctgtacttat tttccaataa aattttcaaa ctttgtactg ttaaaaaaaa


 


5161
aaaaaaaaaa






The amino acid sequence of human IL-1R type 1 isoform 1 precursor, provided by Genbank Accession No. NP_000868.1, is incorporated herein by reference, and is shown below (SEQ ID NO:92).









1
mkvllrlicf iallisslea dkckereeki ilvssaneid vrpcplnpne hkgtitwykd



 


61
dsktpvsteq asrihqhkek lwfvpakved sghyycvvrn ssyclrikis akfvenepnl


 


121
cynaqaifkq klpvagdggl vcpymeffkn ennelpklqw ykdckpllld nihfsgvkdr


 


181
livmnvaekh rgnytchasy tylgkqypit rviefitlee nkptrpvivs panetmevdl


 


241
gsqiqlicnv tgqlsdiayw kwngsvided dpvlgedyys venpankrrs tlitvlnise


 


301
iesrfykhpf tcfaknthgi daayigliyp vtnfqkhmig icvtltviiv csvfiykifk


 


361
idivlwyrds cydflpikas dgktydayil ypktvgegst sdcdifvfkv lpevlekqcg


 


421
yklfiygrdd yvgedivevi nenvkksrrl iiilvretsg fswlggssee qiamynalvq


 


481
dgikvvllel ekiqdyekmp esikfikqkh gairwsgdft qgpqsaktrf wknvryhmpv


 


541
qrrspsskhq llspatkekl qreahvplg


 








(Signal peptide 1-20; mature peptide AA 21-569).







The siRNA used to target human IL-1R type 1 variant 1 mRNA include following sequences (SEQ ID NO: 93-96):









SEQ NO: 93:



5′-UUUCUUCUCACAAACGUGCC-3′



 



SEQ NO: 94:



5′-UUAUACCAAGUUAUAGUGCC-3′



 



SEQ NO: 95:



5′-UUGUAAAACAUCUAAUAGGC-3′



 



SEQ NO: 96:



5′-UUUCCACACUGUAAUAGUCU-3′






The molecular beacon used to target human IL-1R type 1 variant 1 mRNA includes the following sequences (SEQ ID NO: 97-99):









SEQ NO 97:



5′-CCGGTC TTTCTTCTCACAAACGTGC GACCGG-3′



 



SEQ NO 98:



5′-CCGGTC TTAAACACAAAAATATCAC GACCGG-3′



 



SEQ NO 99:



5′-CCGGTC TTTCCACACTGTAATAGTC GACCGG-3′






The mRNA transcript sequence encoding human TNF-alpha, provided by Genbank Accession No. NM_000594.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 100).









1
cagacgctcc ctcagcaagg acagcagagg accagctaag agggagagaa gcaactacag



 


61
accccccctg aaaacaaccc tcagacgcca catcccctga caagctgcca ggcaggttct


 


121
cttcctctca catactgacc cacggctcca ccctctctcc cctggaaagg acaccatgag


 


181
cactgaaagc atgatccggg acgtggagct ggccgaggag gcgctcccca agaagacagg


 


241
ggggccccag ggctccaggc ggtgcttgtt cctcagcctc ttctccttcc tgatcgtggc


 


301
aggcgccacc acgctcttct gcctgctgca ctttggagtg atcggccccc agagggaaga


 


361
gttccccagg gacctctctc taatcagccc tctggcccag gcagtcagat catcttctcg


 


421
aaccccgagt gacaagcctg tagcccatgt tgtagcaaac cctcaagctg aggggcagct


 


481
ccagtggctg aaccgccggg ccaatgccct cctggccaat ggcgtggagc tgagagataa


 


541
ccagctggtg gtgccatcag agggcctgta cctcatctac tcccaggtcc tcttcaaggg


 


601
ccaaggctgc ccctccaccc atgtgctcct cacccacacc atcagccgca tcgccgtctc


 


661
ctaccagacc aaggtcaacc tcctctctgc catcaagagc ccctgccaga gggagacccc


 


721
agagggggct gaggccaagc cctggtatga gcccatctat ctgggagggg tcttccagct


 


781
ggagaagggt gaccgactca gcgctgagat caatcggccc gactatctcg actttgccga


 


841
gtctgggcag gtctactttg ggatcattgc cctgtgagga ggacgaacat ccaaccttcc


 


901
caaacgcctc ccctgcccca atccctttat taccccctcc ttcagacacc ctcaacctct


 


961
tctggctcaa aaagagaatt gggggcttag ggtcggaacc caagcttaga actttaagca


 


1021
acaagaccac cacttcgaaa cctgggattc aggaatgtgt ggcctgcaca gtgaagtgct


 


1081
ggcaaccact aagaattcaa actggggcct ccagaactca ctggggccta cagctttgat


 


1141
ccctgacatc tggaatctgg agaccaggga gcctttggtt ctggccagaa tgctgcagga


 


1201
cttgagaaga cctcacctag aaattgacac aagtggacct taggccttcc tctctccaga


 


1261
tgtttccaga cttccttgag acacggagcc cagccctccc catggagcca gctccctcta


 


1321
tttatgtttg cacttgtgat tatttattat ttatttatta tttatttatt tacagatgaa


 


1381
tgtatttatt tgggagaccg gggtatcctg ggggacccaa tgtaggagct gccttggctc


 


1441
agacatgttt tccgtgaaaa cggagctgaa caataggctg ttcccatgta gccccctggc


 


1501
ctctgtgcct tcttttgatt atgtttttta aaatatttat ctgattaagt tgtctaaaca


 


1561
atgctgattt ggtgaccaac tgtcactcat tgctgagcct ctgctcccca ggggagttgt


 


1621
gtctgtaatc gccctactat tcagtggcga gaaataaagt ttgcttagaa aagaaaaaaa


 


1681
aaaaaa






The amino acid sequence of human TNF-alpha, provided by Genbank Accession No. NP_000585.2, is incorporated herein by reference, and is shown below (SEQ ID NO:101).









1
mstesmirdv elaeealpkk tggpqgsrrc lflslfsfli vagattlfcl lhfgvigpqr



 


61
eefprdlsli splagavrss srtpsdkpva hvvanpqaeg qlqwlnrran allangvelr


 


121
dnqlvvpseg lyliysqvlf kgqgcpsthv llthtisria vsyqtkvnll saikspcqre


 


181
tpegaeakpw yepiylggvf qlekgdrlsa einrpdyldf aesgqvyfgi ial






The siRNA used to target human TNF-alpha mRNA include following sequences (SEQ ID NO: 102-105):









SEQ NO: 102:



5′-AAUAAAUAAUCACAAGUGC-3′



 



SEQ NO: 103:



5′-UAAAAAACAUAAUCAAAAG-3′



 



SEQ NO: 104:



5′-UAAUAAAUAAUCACAAGUG-3′



 



SEQ NO: 105:



5′-UUUUCUUUUCUAAGCAAAC-3′






The molecular beacon used to target human TNF-alpha mRNA includes the following sequences (SEQ ID NO: 106-108):









SEQ NO 106:



5′-CCGGTC AAACATAATCAAAAGAAGG GACCGG-3′



 



SEQ NO 107:



5′-CCGGTC TAAAAAACATAATCAAAAG GACCGG-3′



 



SEQ NO 108:



5′-CCGGTC TATTTTAAAAAACATAATC GACCGG-3′






The mRNA transcript sequence encoding human VEGF A variant 1, provided by Genbank Accession No. NM_001025366.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 109).









1
tcgcggaggc ttggggcagc cgggtagctc ggaggtcgtg gcgctggggg ctagcaccag



 


61
cgctctgtcg ggaggcgcag cggttaggtg gaccggtcag cggactcacc ggccagggcg


 


121
ctcggtgctg gaatttgata ttcattgatc cgggttttat ccctcttctt ttttcttaaa


 


181
catttttttt taaaactgta ttgtttctcg ttttaattta tttttgcttg ccattcccca


 


241
cttgaatcgg gccgacggct tggggagatt gctctacttc cccaaatcac tgtggatttt


 


301
ggaaaccagc agaaagagga aagaggtagc aagagctcca gagagaagtc gaggaagaga


 


361
gagacggggt cagagagagc gcgcgggcgt gcgagcagcg aaagcgacag gggcaaagtg


 


421
agtgacctgc ttttgggggt gaccgccgga gcgcggcgtg agccctcccc cttgggatcc


 


481
cgcagctgac cagtcgcgct gacggacaga cagacagaca ccgcccccag ccccagctac


 


541
cacctcctcc ccggccggcg gcggacagtg gacgcggcgg cgagccgcgg gcaggggccg


 


601
gagcccgcgc ccggaggcgg ggtggagggg gtcggggctc gcggcgtcgc actgaaactt


 


661
ttcgtccaac ttctgggctg ttctcgcttc ggaggagccg tggtccgcgc gggggaagcc


 


721
gagccgagcg gagccgcgag aagtgctagc tcgggccggg aggagccgca gccggaggag


 


781
ggggaggagg aagaagagaa ggaagaggag agggggccgc agtggcgact cggcgctcgg


 


841
aagccgggct catggacggg tgaggcggcg gtgtgcgcag acagtgctcc agccgcgcgc


 


901
gctccccagg ccctggcccg ggcctcgggc cggggaggaa gagtagctcg ccgaggcgcc


 


961
gaggagagcg ggccgcccca cagcccgagc cggagaggga gcgcgagccg cgccggcccc


 


1021
ggtcgggcct ccgaaaccat gaactttctg ctgtcttggg tgcattggag ccttgccttg


 


1081
ctgctctacc tccaccatgc caagtggtcc caggctgcac ccatggcaga aggaggaggg


 


1141
cagaatcatc acgaagtggt gaagttcatg gatgtctatc agcgcagcta ctgccatcca


 


1201
atcgagaccc tggtggacat cttccaggag taccctgatg agatcgagta catcttcaag


 


1261
ccatcctgtg tgcccctgat gcgatgcggg ggctgctgca atgacgaggg cctggagtgt


 


1321
gtgcccactg aggagtccaa catcaccatg cagattatgc ggatcaaacc tcaccaaggc


 


1381
cagcacatag gagagatgag cttcctacag cacaacaaat gtgaatgcag accaaagaaa


 


1441
gatagagcaa gacaagaaaa aaaatcagtt cgaggaaagg gaaaggggca aaaacgaaag


 


1501
cgcaagaaat cccggtataa gtcctggagc gtgtacgttg gtgcccgctg ctgtctaatg


 


1561
ccctggagcc tccctggccc ccatccctgt gggccttgct cagagcggag aaagcatttg


 


1621
tttgtacaag atccgcagac gtgtaaatgt tcctgcaaaa acacagactc gcgttgcaag


 


1681
gcgaggcagc ttgagttaaa cgaacgtact tgcagatgtg acaagccgag gcggtgagcc


 


1741
gggcaggagg aaggagcctc cctcagggtt tcgggaacca gatctctcac caggaaagac


 


1801
tgatacagaa cgatcgatac agaaaccacg ctgccgccac cacaccatca ccatcgacag


 


1861
aacagtcctt aatccagaaa cctgaaatga aggaagagga gactctgcgc agagcacttt


 


1921
gggtccggag ggcgagactc cggcggaagc attcccgggc gggtgaccca gcacggtccc


 


1981
tcttggaatt ggattcgcca ttttattttt cttgctgcta aatcaccgag cccggaagat


 


2041
tagagagttt tatttctggg attcctgtag acacacccac ccacatacat acatttatat


 


2101
atatatatat tatatatata taaaaataaa tatctctatt ttatatatat aaaatatata


 


2161
tattcttttt ttaaattaac agtgctaatg ttattggtgt cttcactgga tgtatttgac


 


2221
tgctgtggac ttgagttggg aggggaatgt tcccactcag atcctgacag ggaagaggag


 


2281
gagatgagag actctggcat gatctttttt ttgtcccact tggtggggcc agggtcctct


 


2341
cccctgccca ggaatgtgca aggccagggc atgggggcaa atatgaccca gttttgggaa


 


2401
caccgacaaa cccagccctg gcgctgagcc tctctacccc aggtcagacg gacagaaaga


 


2461
cagatcacag gtacagggat gaggacaccg gctctgacca ggagtttggg gagcttcagg


 


2521
acattgctgt gctttgggga ttccctccac atgctgcacg cgcatctcgc ccccaggggc


 


2581
actgcctgga agattcagga gcctgggcgg ccttcgctta ctctcacctg cttctgagtt


 


2641
gcccaggaga ccactggcag atgtcccggc gaagagaaga gacacattgt tggaagaagc


 


2701
agcccatgac agctcccctt cctgggactc gccctcatcc tcttcctgct ccccttcctg


 


2761
gggtgcagcc taaaaggacc tatgtcctca caccattgaa accactagtt ctgtcccccc


 


2821
aggagacctg gttgtgtgtg tgtgagtggt tgaccttcct ccatcccctg gtccttccct


 


2881
tcccttcccg aggcacagag agacagggca ggatccacgt gcccattgtg gaggcagaga


 


2941
aaagagaaag tgttttatat acggtactta tttaatatcc ctttttaatt agaaattaaa


 


3001
acagttaatt taattaaaga gtagggtttt ttttcagtat tcttggttaa tatttaattt


 


3061
caactattta tgagatgtat cttttgctct ctcttgctct cttatttgta ccggtttttg


 


3121
tatataaaat tcatgtttcc aatctctctc tccctgatcg gtgacagtca ctagcttatc


 


3181
ttgaacagat atttaatttt gctaacactc agctctgccc tccccgatcc cctggctccc


 


3241
cagcacacat tcctttgaaa taaggtttca atatacatct acatactata tatatatttg


 


3301
gcaacttgta tttgtgtgta tatatatata tatatgttta tgtatatatg tgattctgat


 


3361
aaaatagaca ttgctattct gttttttata tgtaaaaaca aaacaagaaa aaatagagaa


 


3421
ttctacatac taaatctctc tcctttttta attttaatat ttgttatcat ttatttattg


 


3481
gtgctactgt ttatccgtaa taattgtggg gaaaagatat taacatcacg tctttgtctc


 


3541
tagtgcagtt tttcgagata ttccgtagta catatttatt tttaaacaac gacaaagaaa


 


3601
tacagatata tcttaaaaaa aaaaaagcat tttgtattaa agaatttaat tctgatctca


 


3661
aaaaaaaaaa aaaaaaa






The amino acid sequence of human VEGF A isoform 1, provided by Genbank Accession No. NP_001020537.2, is incorporated herein by reference, and is shown below (SEQ ID NO:110).









1
mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg



 


61
csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt


 


121
geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset


 


181
mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd


 


241
ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem


 


301
sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksry kswsvyvgar cclmpwslpg


 


361
phpcgpcser rkhlfvqdpq tckcsckntd srckarqlel nertcrcdkp rr






The siRNA used to target human VEGF Avariant 1 mRNA include following sequences (SEQ ID NO: 111-114):









SEQ NO: 111:



5′-UAAAACUCUCUAAUCUUCCGG-3′



 



SEQ NO: 112:



5′-UUCCUUCUCUUCUUCCUCCUC-3′



 



SEQ NO: 113:



5′-UAUACACACAAAUACAAGUUG-3′



 



SEQ NO: 114:



5′-UUAAAACGAGAAACAAUACAG-3′






The molecular beacon used to target human VEGF Avariant 1 mRNA includes the following sequences (SEQ ID NO: 115-117):









SEQ NO 115:



5′-CCGGTC TAAAACTCTCTAATCTTCC GACCGG-3′



 



SEQ NO 116:



5′-CCGGTC TTTGATCCGCATAATCTGC GACCGG-3′



 



SEQ NO 117:



5′-CCGGTC TTGAAATTAAATATTAACC GACCGG-3′






The mRNA transcript sequence encoding human TGF-beta 1, provided by Genbank Accession No. NM_000660.5, is incorporated herein by reference, and is shown below (SEQ ID NO: 118).









1
agccggtccc cgccgccgcc gcccttcgcg ccctgggcca tctccctccc acctccctcc



 


61
gcggagcagc cagacagcga gggccccggc cgggggcagg ggggacgccc cgtccggggc


 


121
acccccccgg ctctgagccg cccgcggggc cggcctcggc ccggagcgga ggaaggagtc


 


181
gccgaggagc agcctgaggc cccagagtct gagacgagcc gccgccgccc ccgccactgc


 


241
ggggaggagg gggaggagga gcgggaggag ggacgagctg gtcgggagaa gaggaaaaaa


 


301
acttttgaga cttttccgtt gccgctggga gccggaggcg cggggacctc ttggcgcgac


 


361
gctgccccgc gaggaggcag gacttgggga ccccagaccg cctccctttg ccgccgggga


 


421
cgcttgctcc ctccctgccc cctacacggc gtccctcagg cgcccccatt ccggaccagc


 


481
cctcgggagt cgccgacccg gcctcccgca aagacttttc cccagacctc gggcgcaccc


 


541
cctgcacgcc gccttcatcc ccggcctgtc tcctgagccc ccgcgcatcc tagacccttt


 


601
ctcctccagg agacggatct ctctccgacc tgccacagat cccctattca agaccaccca


 


661
ccttctggta ccagatcgcg cccatctagg ttatttccgt gggatactga gacacccccg


 


721
gtccaagcct cccctccacc actgcgccct tctccctgag gacctcagct ttccctcgag


 


781
gccctcctac cttttgccgg gagaccccca gcccctgcag gggcggggcc tccccaccac


 


841
accagccctg ttcgcgctct cggcagtgcc ggggggcgcc gcctccccca tgccgccctc


 


901
cgggctgcgg ctgctgccgc tgctgctacc gctgctgtgg ctactggtgc tgacgcctgg


 


961
ccggccggcc gcgggactat ccacctgcaa gactatcgac atggagctgg tgaagcggaa


 


1021
gcgcatcgag gccatccgcg gccagatcct gtccaagctg cggctcgcca gccccccgag


 


1081
ccagggggag gtgccgcccg gcccgctgcc cgaggccgtg ctcgccctgt acaacagcac


 


1141
ccgcgaccgg gtggccgggg agagtgcaga accggagccc gagcctgagg ccgactacta


 


1201
cgccaaggag gtcacccgcg tgctaatggt ggaaacccac aacgaaatct atgacaagtt


 


1261
caagcagagt acacacagca tatatatgtt cttcaacaca tcagagctcc gagaagcggt


 


1321
acctgaaccc gtgttgctct cccgggcaga gctgcgtctg ctgaggctca agttaaaagt


 


1381
ggagcagcac gtggagctgt accagaaata cagcaacaat tcctggcgat acctcagcaa


 


1441
ccggctgctg gcacccagcg actcgccaga gtggttatct tttgatgtca ccggagttgt


 


1501
gcggcagtgg ttgagccgtg gaggggaaat tgagggcttt cgccttagcg cccactgctc


 


1561
ctgtgacagc agggataaca cactgcaagt ggacatcaac gggttcacta ccggccgccg


 


1621
aggtgacctg gccaccattc atggcatgaa ccggcctttc ctgcttctca tggccacccc


 


1681
gctggagagg gcccagcatc tgcaaagctc ccggcaccgc cgagccctgg acaccaacta


 


1741
ttgcttcagc tccacggaga agaactgctg cgtgcggcag ctgtacattg acttccgcaa


 


1801
ggacctcggc tggaagtgga tccacgagcc caagggctac catgccaact tctgcctcgg


 


1861
gccctgcccc tacatttgga gcctggacac gcagtacagc aaggtcctgg ccctgtacaa


 


1921
ccagcataac ccgggcgcct cggcggcgcc gtgctgcgtg ccgcaggcgc tggagccgct


 


1981
gcccatcgtg tactacgtgg gccgcaagcc caaggtggag cagctgtcca acatgatcgt


 


2041
gcgctcctgc aagtgcagct gaggtcccgc cccgccccgc cccgccccgg caggcccggc


 


2101
cccaccccgc cccgcccccg ctgccttgcc catgggggct gtatttaagg acacccgtgc


 


2161
cccaagccca cctggggccc cattaaagat ggagagagga ctgcggatct ctgtgtcatt


 


2221
gggcgcctgc ctggggtctc catccctgac gttcccccac tcccactccc tctctctccc


 


2281
tctctgcctc ctcctgcctg tctgcactat tcctttgccc ggcatcaagg cacaggggac


 


2341
cagtggggaa cactactgta gttagatcta tttattgagc accttgggca ctgttgaagt


 


2401
gccttacatt aatgaactca ttcagtcacc atagcaacac tctgagatgc agggactctg


 


2461
ataacaccca ttttaaaggt gaggaaacaa gcccagagag gttaagggag gagttcctgc


 


2521
ccaccaggaa cctgctttag tgggggatag tgaagaagac aataaaagat agtagttcag


 


2581
gcc






The amino acid sequence of human TGF-beta 1 (precursor), provided by Genbank Accession No. NP_000651.3, is incorporated herein by reference, and is shown below (SEQ ID NO:119).









1
mppsglrllp lllpllwllv ltpgrpaagl stcktidmel vkrkrieair gqilsklrla



 


61
sppsqgevpp gplpeavlal ynstrdrvag esaepepepe adyyakevtr vlmvethnei


 


121
ydkfkqsths iymffntsel reavpepvll sraelrllrl klkveqhvel yqkysnnswr


 


181
ylsnrllaps dspewlsfdv tgvvrqwlsr ggeiegfrls ahcscdsrdn tlqvdingft


 


241
tgrrgdlati hgmnrpflll matpleraqh lqssrhrral dtnycfsste knccvrqlyi


 


301
dfrkdlgwkw ihepkgyhan fclgpcpyiw sldtqyskvl alynqhnpga saapccvpqa


 


361
leplpivyyv grkpkveqls nmivrsckcs


 








(Signal peptide AA 1-29; mature peptide AA 30-278).







The siRNA used to target human TGF-beta 1 mRNA include following sequences (SEQ ID NO: 120-123):









SEQ NO: 120:



5′-UAUUGUCUUCUUCACUAUC-3′



 



SEQ NO: 121:



5′-UAGAUCUAACUACAGUAGU-3′



 



SEQ NO: 122:



5′-UAUAUGCUGUGUGUACUCU-3′



 



SEQ NO: 123:



5′-UAUAUAUGCUGUGUGUACU-3′






The molecular beacon used to target human TGF-beta 1 mRNA includes the following sequences (SEQ ID NO: 124-126):









SEQ NO 124:



5′-CCGGTC ATATATGCTGTGTGTACTC GACCGG-3′


 



SEQ NO 125:



5′-CCGGTC TTTTATTGTCTTCTTCACT GACCGG-3′


 



SEQ NO 126:



5′-CCGGTC TATATATGCTGTGTGTACT GACCGG-3′






The mRNA transcript sequence encoding human TGF-beta 2 variant 1, provided by Genbank Accession No. NM_001135599.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 127).









1
gtgatgttat ctgctggcag cagaaggttc gctccgagcg gagctccaga agctcctgac



 


61
aagagaaaga cagattgaga tagagataga aagagaaaga gagaaagaga cagcagagcg


 


121
agagcgcaag tgaaagaggc aggggagggg gatggagaat attagcctga cggtctaggg


 


181
agtcatccag gaacaaactg aggggctgcc cggctgcaga caggaggaga cagagaggat


 


241
ctattttagg gtggcaagtg cctacctacc ctaagcgagc aattccacgt tggggagaag


 


301
ccagcagagg ttgggaaagg gtgggagtcc aagggagccc ctgcgcaacc ccctcaggaa


 


361
taaaactccc cagccagggt gtcgcaaggg ctgccgttgt gatccgcagg gggtgaacgc


 


421
aaccgcgacg gctgatcgtc tgtggctggg ttggcgtttg gagcaagaga aggaggagca


 


481
ggagaaggag ggagctggag gctggaagcg tttgcaagcg gcggcggcag caacgtggag


 


541
taaccaagcg ggtcagcgcg cgcccgccag ggtgtaggcc acggagcgca gctcccagag


 


601
caggatccgc gccgcctcag cagcctctgc ggcccctgcg gcacccgacc gagtaccgag


 


661
cgccctgcga agcgcaccct cctccccgcg gtgcgctggg ctcgccccca gcgcgcgcac


 


721
acgcacacac acacacacac acacacacgc acgcacacac gtgtgcgctt ctctgctccg


 


781
gagctgctgc tgctcctgct ctcagcgccg cagtggaagg caggaccgaa ccgctccttc


 


841
tttaaatata taaatttcag cccaggtcag cctcggcggc ccccctcacc gcgctcccgg


 


901
cgcccctccc gtcagttcgc cagctgccag ccccgggacc ttttcatctc ttcccttttg


 


961
gccggaggag ccgagttcag atccgccact ccgcacccga gactgacaca ctgaactcca


 


1021
cttcctcctc ttaaatttat ttctacttaa tagccactcg tctctttttt tccccatctc


 


1081
attgctccaa gaattttttt cttcttactc gccaaagtca gggttccctc tgcccgtccc


 


1141
gtattaatat ttccactttt ggaactactg gccttttctt tttaaaggaa ttcaagcagg


 


1201
atacgttttt ctgttgggca ttgactagat tgtttgcaaa agtttcgcat caaaaacaac


 


1261
aacaacaaaa aaccaaacaa ctctccttga tctatacttt gagaattgtt gatttctttt


 


1321
ttttattctg acttttaaaa acaacttttt tttccacttt tttaaaaaat gcactactgt


 


1381
gtgctgagcg cttttctgat cctgcatctg gtcacggtcg cgctcagcct gtctacctgc


 


1441
agcacactcg atatggacca gttcatgcgc aagaggatcg aggcgatccg cgggcagatc


 


1501
ctgagcaagc tgaagctcac cagtccccca gaagactatc ctgagcccga ggaagtcccc


 


1561
ccggaggtga tttccatcta caacagcacc agggacttgc tccaggagaa ggcgagccgg


 


1621
agggcggccg cctgcgagcg cgagaggagc gacgaagagt actacgccaa ggaggtttac


 


1681
aaaatagaca tgccgccctt cttcccctcc gaaactgtct gcccagttgt tacaacaccc


 


1741
tctggctcag tgggcagctt gtgctccaga cagtcccagg tgctctgtgg gtaccttgat


 


1801
gccatcccgc ccactttcta cagaccctac ttcagaattg ttcgatttga cgtctcagca


 


1861
atggagaaga atgcttccaa tttggtgaaa gcagagttca gagtctttcg tttgcagaac


 


1921
ccaaaagcca gagtgcctga acaacggatt gagctatatc agattctcaa gtccaaagat


 


1981
ttaacatctc caacccagcg ctacatcgac agcaaagttg tgaaaacaag agcagaaggc


 


2041
gaatggctct ccttcgatgt aactgatgct gttcatgaat ggcttcacca taaagacagg


 


2101
aacctgggat ttaaaataag cttacactgt ccctgctgca cttttgtacc atctaataat


 


2161
tacatcatcc caaataaaag tgaagaacta gaagcaagat ttgcaggtat tgatggcacc


 


2221
tccacatata ccagtggtga tcagaaaact ataaagtcca ctaggaaaaa aaacagtggg


 


2281
aagaccccac atctcctgct aatgttattg ccctcctaca gacttgagtc acaacagacc


 


2341
aaccggcgga agaagcgtgc tttggatgcg gcctattgct ttagaaatgt gcaggataat


 


2401
tgctgcctac gtccacttta cattgatttc aagagggatc tagggtggaa atggatacac


 


2461
gaacccaaag ggtacaatgc caacttctgt gctggagcat gcccgtattt atggagttca


 


2521
gacactcagc acagcagggt cctgagctta tataatacca taaatccaga agcatctgct


 


2581
tctccttgct gcgtgtccca agatttagaa cctctaacca ttctctacta cattggcaaa


 


2641
acacccaaga ttgaacagct ttctaatatg attgtaaagt cttgcaaatg cagctaaaat


 


2701
tcttggaaaa gtggcaagac caaaatgaca atgatgatga taatgatgat gacgacgaca


 


2761
acgatgatgc ttgtaacaag aaaacataag agagccttgg ttcatcagtg ttaaaaaatt


 


2821
tttgaaaagg cggtactagt tcagacactt tggaagtttg tgttctgttt gttaaaactg


 


2881
gcatctgaca caaaaaaagt tgaaggcctt attctacatt tcacctactt tgtaagtgag


 


2941
agagacaaga agcaaatttt ttttaaagaa aaaaataaac actggaagaa tttattagtg


 


3001
ttaattatgt gaacaacgac aacaacaaca acaacaacaa acaggaaaat cccattaagt


 


3061
ggagttgctg tacgtaccgt tcctatcccg cgcctcactt gatttttctg tattgctatg


 


3121
caataggcac ccttcccatt cttactctta gagttaacag tgagttattt attgtgtgtt


 


3181
actatataat gaacgtttca ttgcccttgg aaaataaaac aggtgtataa agtggagacc


 


3241
aaatactttg ccagaaactc atggatggct taaggaactt gaactcaaac gagccagaaa


 


3301
aaaagaggtc atattaatgg gatgaaaacc caagtgagtt attatatgac cgagaaagtc


 


3361
tgcattaaga taaagaccct gaaaacacat gttatgtatc agctgcctaa ggaagcttct


 


3421
tgtaaggtcc aaaaactaaa aagactgtta ataaaagaaa ctttcagtca gaataagtct


 


3481
gtaagttttt ttttttcttt ttaattgtaa atggttcttt gtcagtttag taaaccagtg


 


3541
aaatgttgaa atgttttgac atgtactggt caaacttcag accttaaaat attgctgtat


 


3601
agctatgcta taggtttttt cctttgtttt ggtatatgta accataccta tattattaaa


 


3661
atagatggat atagaagcca gcataattga aaacacatct gcagatctct tttgcaaact


 


3721
attaaatcaa aacattaact actttatgtg taatgtgtaa atttttacca tattttttat


 


3781
attctgtaat aatgtcaact atgatttaga ttgacttaaa tttgggctct ttttaatgat


 


3841
cactcacaaa tgtatgtttc ttttagctgg ccagtacttt tgagtaaagc ccctatagtt


 


3901
tgacttgcac tacaaatgca tttttttttt aataacattt gccctacttg tgctttgtgt


 


3961
ttctttcatt attatgacat aagctacctg ggtccacttg tcttttcttt tttttgtttc


 


4021
acagaaaaga tgggttcgag ttcagtggtc ttcatcttcc aagcatcatt actaaccaag


 


4081
tcagacgtta acaaattttt atgttaggaa aaggaggaat gttatagata catagaaaat


 


4141
tgaagtaaaa tgttttcatt ttagcaagga tttagggttc taactaaaac tcagaatctt


 


4201
tattgagtta agaaaagttt ctctaccttg gtttaatcaa tatttttgta aaatcctatt


 


4261
gttattacaa agaggacact tcataggaaa catctttttc tttagtcagg tttttaatat


 


4321
tcagggggaa attgaaagat atatatttta gtcgattttt caaaagggga aaaaagtcca


 


4381
ggtcagcata agtcattttg tgtatttcac tgaagttata aggtttttat aaatgttctt


 


4441
tgaaggggaa aaggcacaag ccaatttttc ctatgatcaa aaaattcttt ctttcctctg


 


4501
agtgagagtt atctatatct gaggctaaag tttaccttgc tttaataaat aatttgccac


 


4561
atcattgcag aagaggtatc ctcatgctgg ggttaataga atatgtcagt ttatcacttg


 


4621
tcgcttattt agctttaaaa taaaaattaa taggcaaagc aatggaatat ttgcagtttc


 


4681
acctaaagag cagcataagg aggcgggaat ccaaagtgaa gttgtttgat atggtctact


 


4741
tcttttttgg aatttcctga ccattaatta aagaattgga tttgcaagtt tgaaaactgg


 


4801
aaaagcaaga gatgggatgc cataatagta aacagccctt gtgttggatg taacccaatc


 


4861
ccagatttga gtgtgtgttg attatttttt tgtcttccac ttttctatta tgtgtaaatc


 


4921
acttttattt ctgcagacat tttcctctca gataggatga cattttgttt tgtattattt


 


4981
tgtctttcct catgaatgca ctgataatat tttaaatgct ctattttaag atctcttgaa


 


5041
tctgtttttt ttttttttaa tttgggggtt ctgtaaggtc tttatttccc ataagtaaat


 


5101
attgccatgg gaggggggtg gaggtggcaa ggaaggggtg aagtgctagt atgcaagtgg


 


5161
gcagcaatta tttttgtgtt aatcagcagt acaatttgat cgttggcatg gttaaaaaat


 


5221
ggaatataag attagctgtt ttgtattttg atgaccaatt acgctgtatt ttaacacgat


 


5281
gtatgtctgt ttttgtggtg ctctagtggt aaataaatta tttcgatgat atgtggatgt


 


5341
ctttttccta tcagtaccat catcgagtct agaaaacacc tgtgatgcaa taagactatc


 


5401
tcaagctgga aaagtcatac cacctttccg attgccctct gtgctttctc ccttaaggac


 


5461
agtcacttca gaagtcatgc tttaaagcac aagagtcagg ccatatccat caaggataga


 


5521
agaaatccct gtgccgtctt tttattccct tatttattgc tatttggtaa ttgtttgaga


 


5581
tttagtttcc atccagcttg actgccgacc agaaaaaatg cagagagatg tttgcaccat


 


5641
gctttggctt tctggttcta tgttctgcca acgccagggc caaaagaact ggtctagaca


 


5701
gtatcccctg tagccccata acttggatag ttgctgagcc agccagatat aacaagagcc


 


5761
acgtgctttc tggggttggt tgtttgggat cagctacttg cctgtcagtt tcactggtac


 


5821
cactgcacca caaacaaaaa aacccaccct atttcctcca atttttttgg ctgctaccta


 


5881
caagaccaga ctcctcaaac gagttgccaa tctcttaata aataggatta ataaaaaaag


 


5941
taattgtgac tcaaaaaaaa aaaaaa






The amino acid sequence of human TGF-beta 2 isoform 1 precursor, provided by Genbank Accession No. NP_001129071.1, is incorporated herein by reference, and is shown below (SEQ ID NO:128).









1
mhycvlsafl ilhlvtvals lstcstldmd qfmrkrieai rgqilsklkl tsppedypep



 


61
eevppevisi ynstrdllqe kasrraaace rersdeeyya kevykidmpp ffpsetvcpv


 


121
vttpsgsvgs lcsrqsqvlc gyldaipptf yrpyfrivrf dvsameknas nlvkaefrvf


 


181
rlqnpkarvp eqrielyqil kskdltsptq ryidskvvkt raegewlsfd vtdavhewlh


 


241
hkdrnlgfki slhcpcctfv psnnyiipnk seelearfag idgtstytsg dqktikstrk


 


301
knsgktphll lmllpsyrle sqqtnrrkkr aldaaycfrn vqdncclrpl yidfkrdlgw


 


361
kwihepkgyn anfcagacpy lwssdtqhsr vlslyntinp easaspccvs qdlepltily


 


421
yigktpkieq lsnmivksck cs






The siRNA used to target human TGF-beta 2 variant 1 mRNA include following sequences (SEQ ID NO: 129-132):









SEQ NO: 129:



5′-UAUCUCUAUCUCAAUCUGUC-3′


 



SEQ NO: 130:



5′-UUCUAUCUCUAUCUCAAUCU-3′


 



SEQ NO: 131:



5′-UUCUCUUUCUAUCUCUAUCU-3′


 



SEQ NO: 132:



5′-UCUAUCUCUAUCUCAAUCUG-3′






The molecular beacon used to target human TGF-beta 2 variant 1 mRNA includes the following sequences (SEQ ID NO: 133-135):









SEQ NO 133:



5′-CCGGTC TTCTATCTCTATCTCAATC GACCGG-3′


 



SEQ NO 134:



5′-CCGGTC TATCTCTATCTCAATCTGT GACCGG-3′


 



SEQ NO 135:



5′-CCGGTC TTCTCTTTCTATCTCTATC GACCGG-3′






The mRNA transcript sequence encoding human IGF-1 variant 4, provided by Genbank Accession No. NM_000618.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 136).









1
ttttgtagat aaatgtgagg attttctcta aatccctctt ctgtttgcta aatctcactg



 


61
tcactgctaa attcagagca gatagagcct gcgcaatgga ataaagtcct caaaattgaa


 


121
atgtgacatt gctctcaaca tctcccatct ctctggattt ctttttgctt cattattcct


 


181
gctaaccaat tcattttcag actttgtact tcagaagcaa tgggaaaaat cagcagtctt


 


241
ccaacccaat tatttaagtg ctgcttttgt gatttcttga aggtgaagat gcacaccatg


 


301
tcctcctcgc atctcttcta cctggcgctg tgcctgctca ccttcaccag ctctgccacg


 


361
gctggaccgg agacgctctg cggggctgag ctggtggatg ctcttcagtt cgtgtgtgga


 


421
gacaggggct tttatttcaa caagcccaca gggtatggct ccagcagtcg gagggcgcct


 


481
cagacaggca tcgtggatga gtgctgcttc cggagctgtg atctaaggag gctggagatg


 


541
tattgcgcac ccctcaagcc tgccaagtca gctcgctctg tccgtgccca gcgccacacc


 


601
gacatgccca agacccagaa ggaagtacat ttgaagaacg caagtagagg gagtgcagga


 


661
aacaagaact acaggatgta ggaagaccct cctgaggagt gaagagtgac atgccaccgc


 


721
aggatccttt gctctgcacg agttacctgt taaactttgg aacacctacc aaaaaataag


 


781
tttgataaca tttaaaagat gggcgtttcc cccaatgaaa tacacaagta aacattccaa


 


841
cattgtcttt aggagtgatt tgcaccttgc aaaaatggtc ctggagttgg tagattgctg


 


901
ttgatctttt atcaataatg ttctatagaa aagaaaaaaa aaatatatat atatatatat


 


961
cttagtccct gcctctcaag agccacaaat gcatgggtgt tgtatagatc cagttgcact


 


1021
aaattcctct ctgaatcttg gctgctggag ccattcattc agcaaccttg tctaagtggt


 


1081
ttatgaattg tttccttatt tgcacttctt tctacacaac tcgggctgtt tgttttacag


 


1141
tgtctgataa tcttgttagt ctatacccac cacctccctt cataaccttt atatttgccg


 


1201
aatttggcct cctcaaaagc agcagcaagt cgtcaagaag cacaccaatt ctaacccaca


 


1261
agattccatc tgtggcattt gtaccaaata taagttggat gcattttatt ttagacacaa


 


1321
agctttattt ttccacatca tgcttacaaa aaagaataat gcaaatagtt gcaactttga


 


1381
ggccaatcat ttttaggcat atgttttaaa catagaaagt ttcttcaact caaaagagtt


 


1441
ccttcaaatg atgagttaat gtgcaaccta attagtaact ttcctctttt tattttttcc


 


1501
atatagagca ctatgtaaat ttagcatatc aattatacag gatatatcaa acagtatgta


 


1561
aaactctgtt ttttagtata atggtgctat tttgtagttt gttatatgaa agagtctggc


 


1621
caaaacggta atacgtgaaa gcaaaacaat aggggaagcc tggagccaaa gatgacacaa


 


1681
ggggaagggt actgaaaaca ccatccattt gggaaagaag gcaaagtccc cccagttatg


 


1741
ccttccaaga ggaacttcag acacaaaagt ccactgatgc aaattggact ggcgagtcca


 


1801
gagaggaaac tgtggaatgg aaaaagcaga aggctaggaa ttttagcagt cctggtttct


 


1861
ttttctcatg gaagaaatga acatctgcca gctgtgtcat ggactcacca ctgtgtgacc


 


1921
ttgggcaagt cacttcacct ctctgtgcct cagtttcctc atctgcaaaa tgggggcaat


 


1981
atgtcatcta cctacctcaa aggggtggta taaggtttaa aaagataaag attcagattt


 


2041
tttttaccct gggttgctgt aagggtgcaa catcagggcg cttgagttgc tgagatgcaa


 


2101
ggaattctat aaataaccca ttcatagcat agctagagat tggtgaattg aatgctcctg


 


2161
acatctcagt tcttgtcagt gaagctatcc aaataactgg ccaactagtt gttaaaagct


 


2221
aacagctcaa tctcttaaaa cacttttcaa aatatgtggg aagcatttga ttttcaattt


 


2281
gattttgaat tctgcatttg gttttatgaa tacaaagata agtgaaaaga gagaaaggaa


 


2341
aagaaaaagg agaaaaacaa agagatttct accagtgaaa ggggaattaa ttactctttg


 


2401
ttagcactca ctgactcttc tatgcagtta ctacatatct agtaaaacct cgtttaatac


 


2461
tataaataat attctattca ttttgaaaaa cacaatgatt ccttcttttc taggcaatat


 


2521
aaggaaagtg atccaaaatt tgaaatatta aaataatatc taataaaaag tcacaaagtt


 


2581
atcttcttta acaaacttta ctcttattct tagctgtata tacatttttt taaaagtttg


 


2641
ttaaaatatg cttgactaga gtttccagtt gaaaggcaaa aacttccatc acaacaagaa


 


2701
atttcccatg cctgctcaga agggtagccc ctagctctct gtgaatgtgt tttatccatt


 


2761
caactgaaaa ttggtatcaa gaaagtccac tggttagtgt actagtccat catagcctag


 


2821
aaaatgatcc ctatctgcag atcaagattt tctcattaga acaatgaatt atccagcatt


 


2881
cagatctttc tagtcacctt agaacttttt ggttaaaagt acccaggctt gattatttca


 


2941
tgcaaattct atattttaca ttcttggaaa gtctatatga aaaacaaaaa taacatcttc


 


3001
agtttttctc ccactgggtc acctcaagga tcagaggcca ggaaaaaaaa aaaaaagact


 


3061
ccctggatct ctgaatatat gcaaaaagaa ggccccattt agtggagcca gcaatcctgt


 


3121
tcagtcaaca agtattttaa ctctcagtcc aacattattt gaattgagca cctcaagcat


 


3181
gcttagcaat gttctaatca ctatggacag atgtaaaaga aactatacat catttttgcc


 


3241
ctctgcctgt tttccagaca tacaggttct gtggaataag atactggact cctcttccca


 


3301
agatggcact tctttttatt tcttgtcccc agtgtgtacc ttttaaaatt attccctctc


 


3361
aacaaaactt tataggcagt cttctgcaga cttaacgtgt tttctgtcat agttagatgt


 


3421
gataattcta agagtgtcta tgacttattt ccttcactta attctatcca cagtcaaaaa


 


3481
tcccccaagg aggaaagctg aaagatgcac tgccatatta tctttcttaa ctttttccaa


 


3541
cacataatcc tctccaactg gattataaat aaattgaaaa taactcatta taccaattca


 


3601
ctattttatt ttttaatgaa ttaaaactag aaaacaaatt gatgcaaacc ctggaagtca


 


3661
gttgattact atatactaca gcagaatgac tcagatttca tagaaaggag caaccaaaat


 


3721
gtcacaaccc aaaactttac aagctttgct tcagaattag attgctttat aattcttgaa


 


3781
tgaggcaatt tcaagatatt tgtaaaagaa cagtaaacat tggtaagaat gagctttcaa


 


3841
ctcataggct tatttccaat ttaattgacc atactggata cttaggtcaa atttctgttc


 


3901
tctcttcccc aaataatatt aaagtattat ttgaactttt taagatgagg cagttcccct


 


3961
gaaaaagtta atgcagctct ccatcagaat ccactcttct agggatatga aaatctctta


 


4021
acacccaccc tacatacaca gacacacaca cacacacaca cacacacaca cacacacaca


 


4081
ttcaccctaa ggatccaatg gaatactgaa aagaaatcac ttccttgaaa attttattaa


 


4141
aaaacaaaca aacaaacaaa aagcctgtcc acccttgaga atccttcctc tccttggaac


 


4201
gtcaatgttt gtgtagatga aaccatctca tgctctgtgg ctccagggtt tctgttacta


 


4261
ttttatgcac ttgggagaag gcttagaata aaagatgtag cacattttgc tttcccattt


 


4321
attgtttggc cagctatgcc aatgtggtgc tattgtttct ttaagaaagt acttgactaa


 


4381
aaaaaaaaga aaaaaagaaa aaaaagaaag catagacata tttttttaaa gtataaaaac


 


4441
aacaattcta tagatagatg gcttaataaa atagcattag gtctatctag ccaccaccac


 


4501
ctttcaactt tttatcactc acaagtagtg tactgttcac caaattgtga atttgggggt


 


4561
gcaggggcag gagttggaaa ttttttaaag ttagaaggct ccattgtttt gttggctctc


 


4621
aaacttagca aaattagcaa tatattatcc aatcttctga acttgatcaa gagcatggag


 


4681
aataaacgcg ggaaaaaaga tcttataggc aaatagaaga atttaaaaga taagtaagtt


 


4741
ccttattgat ttttgtgcac tctgctctaa aacagatatt cagcaagtgg agaaaataag


 


4801
aacaaagaga aaaaatacat agatttacct gcaaaaaata gcttctgcca aatccccctt


 


4861
gggtattctt tggcatttac tggtttatag aagacattct cccttcaccc agacatctca


 


4921
aagagcagta gctctcatga aaagcaatca ctgatctcat ttgggaaatg ttggaaagta


 


4981
tttccttatg agatgggggt tatctactga taaagaaaga atttatgaga aattgttgaa


 


5041
agagatggct aacaatctgt gaagattttt tgtttcttgt ttttgttttt tttttttttt


 


5101
tactttatac agtctttatg aatttcttaa tgttcaaaat gacttggttc ttttcttctt


 


5161
tttttatatc agaatgagga ataataagtt aaacccacat agactcttta aaactatagg


 


5221
ctagatagaa atgtatgttt gacttgttga agctataatc agactattta aaatgttttg


 


5281
ctatttttaa tcttaaaaga ttgtgctaat ttattagagc agaacctgtt tggctctcct


 


5341
cagaagaaag aatctttcca ttcaaatcac atggctttcc accaatattt tcaaaagata


 


5401
aatctgattt atgcaatggc atcatttatt ttaaaacaga agaattgtga aagtttatgc


 


5461
ccctcccttg caaagaccat aaagtccaga tctggtaggg gggcaacaac aaaaggaaaa


 


5521
tgttgttgat tcttggtttt ggattttgtt ttgttttcaa tgctagtgtt taatcctgta


 


5581
gtacatattt gcttattgct attttaatat tttataagac cttcctgtta ggtattagaa


 


5641
agtgatacat agatatcttt tttgtgtaat ttctatttaa aaaagagaga agactgtcag


 


5701
aagctttaag tgcatatggt acaggataaa gatatcaatt taaataacca attcctatct


 


5761
ggaacaatgc ttttgttttt taaagaaacc tctcacagat aagacagagg cccaggggat


 


5821
ttttgaagct gtctttattc tgcccccatc ccaacccagc ccttattatt ttagtatctg


 


5881
cctcagaatt ttatagaggg ctgaccaagc tgaaactcta gaattaaagg aacctcactg


 


5941
aaaacatata tttcacgtgt tccctctttt tttttttcct ttttgtgaga tggggtctcg


 


6001
cactgtcccc caggctggag tgcagtggca tgatctcggc tcactgcaac ctccacctcc


 


6061
tgggtttaag cgattctcct gcctcagcct cctgagtagc tgggattaca ggcacccacc


 


6121
actatgcccg gctaattttt tggattttta atagagacgg ggttttacca tgttggccag


 


6181
gttggtctca aactcctgac cttgtgattt gcccgcctca gcctcccaaa ttgctgggat


 


6241
tacaggcatg agccaccaca ccctgcccat gtgttccctc ttaatgtatg attacatgga


 


6301
tcttaaacat gatccttctc tcctcattct tcaactatct ttgatggggt ctttcaaggg


 


6361
gaaaaaaatc caagcttttt taaagtaaaa aaaaaaaaag agaggacaca aaaccaaatg


 


6421
ttactgctca actgaaatat gagttaagat ggagacagag tttctcctaa taaccggagc


 


6481
tgaattacct ttcactttca aaaacatgac cttccacaat ccttagaatc tgcctttttt


 


6541
tatattactg aggcctaaaa gtaaacatta ctcattttat tttgcccaaa atgcactgat


 


6601
gtaaagtagg aaaaataaaa acagagctct aaaatccctt tcaagccacc cattgacccc


 


6661
actcaccaac tcatagcaaa gtcacttctg ttaatccctt aatctgattt tgtttggata


 


6721
tttatcttgt acccgctgct aaacacactg caggagggac tctgaaacct caagctgtct


 


6781
acttacatct tttatctgtg tctgtgtatc atgaaaatgt ctattcaaaa tatcaaaacc


 


6841
tttcaaatat cacgcagctt atattcagtt tacataaagg ccccaaatac catgtcagat


 


6901
ctttttggta aaagagttaa tgaactatga gaattgggat tacatcatgt attttgcctc


 


6961
atgtattttt atcacactta taggccaagt gtgataaata aacttacaga cactgaatta


 


7021
atttcccctg ctactttgaa accagaaaat aatgactggc cattcgttac atctgtctta


 


7081
gttgaaaagc atatttttta ttaaattaat tctgattgta tttgaaatta ttattcaatt


 


7141
cacttatggc agaggaatat caatcctaat gacttctaaa aatgtaacta attgaatcat


 


7201
tatcttacat ttactgttta ataagcatat tttgaaaatg tatggctaga gtgtcataat


 


7261
aaaatggtat atctttcttt agtaattaca ttaaaattag tcatgtttga ttaattagtt


 


7321
c






The amino acid sequence of human IGF-1 isoform 4 preproprotein, provided by Genbank Accession No. NP_000609.1, is incorporated herein by reference, and is shown below (SEQ ID NO:137).









1
mgkisslptq lfkccfcdfl kvkmhtmsss hlfylalcll tftssatagp etlcgaelvd



 


61
alqfvcgdrg fyfnkptgyg sssrrapqtg ivdeccfrsc dlrrlemyca plkpaksars


 


121
vragrhtdmp ktgkevhlkn asrgsagnkn yrm






The siRNA used to target human IGF-1 variant 4 mRNA include following sequences (SEQ ID NO: 138-141):









SEQ NO: 138:



5′-UAAACUGAAUAUAAGCUGC-3′


 



SEQ NO: 139:



5′-UAAAAAAAUAUGUCUAUGC-3′


 



SEQ NO: 140:



5′-UUUAACAGGUAACUCGUGC-3′


 



SEQ NO: 141:



5′-UAACAAACUACAAAAUAGC-3′






The molecular beacon used to target human IGF-1 variant 4 mRNA includes the following sequences (SEQ ID NO: 142-144):









SEQ NO 142:



5′-CCGGTC TAAACTGAATATAAGCTGCG GACCGG-3′



 



SEQ NO 143:



5′-CCGGTC TTTAAATTCTTCTATTTGCC GACCGG-3′



 



SEQ NO 144:



5′-CCGGTC TAATCAACTGACTTCCAGGGGACCGG-3′






The mRNA transcript sequence encoding human BMP-2, provided by Genbank Accession No. NM_001200.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 145).









1
ccacaaaggg cacttggccc cagggctagg agagcgaggg gagagcacag ccacccgcct



 


61
cggcggcccg ggactcggct cgactcgccg gagaatgcgc ccgaggacga cggggcgcca


 


121
gagccgcggt gctttcaact ggcgagcgcg aatgggggtg cactggagta aggcagagtg


 


181
atgcgggggg gcaactcgcc tggcaccgag atcgccgccg tgcccttccc tggacccggc


 


241
gtcgcccagg atggctgccc cgagccatgg gccgcggcgg agctagcgcg gagcgcccga


 


301
ccctcgaccc ccgagtcccg gagccggccc cgcgcggggc cacgcgtccc tcgggcgctg


 


361
gttcctaagg aggacgacag caccagcttc tcctttctcc cttcccttcc ctgccccgca


 


421
ctcctccccc tgctcgctgt tgttgtgtgt cagcacttgg ctggggactt cttgaacttg


 


481
cagggagaat aacttgcgca ccccactttg cgccggtgcc tttgccccag cggagcctgc


 


541
ttcgccatct ccgagcccca ccgcccctcc actcctcggc cttgcccgac actgagacgc


 


601
tgttcccagc gtgaaaagag agactgcgcg gccggcaccc gggagaagga ggaggcaaag


 


661
aaaaggaacg gacattcggt ccttgcgcca ggtcctttga ccagagtttt tccatgtgga


 


721
cgctctttca atggacgtgt ccccgcgtgc ttcttagacg gactgcggtc tcctaaaggt


 


781
cgaccatggt ggccgggacc cgctgtcttc tagcgttgct gcttccccag gtcctcctgg


 


841
gcggcgcggc tggcctcgtt ccggagctgg gccgcaggaa gttcgcggcg gcgtcgtcgg


 


901
gccgcccctc atcccagccc tctgacgagg tcctgagcga gttcgagttg cggctgctca


 


961
gcatgttcgg cctgaaacag agacccaccc ccagcaggga cgccgtggtg cccccctaca


 


1021
tgctagacct gtatcgcagg cactcaggtc agccgggctc acccgcccca gaccaccggt


 


1081
tggagagggc agccagccga gccaacactg tgcgcagctt ccaccatgaa gaatctttgg


 


1141
aagaactacc agaaacgagt gggaaaacaa cccggagatt cttctttaat ttaagttcta


 


1201
tccccacgga ggagtttatc acctcagcag agcttcaggt tttccgagaa cagatgcaag


 


1261
atgctttagg aaacaatagc agtttccatc accgaattaa tatttatgaa atcataaaac


 


1321
ctgcaacagc caactcgaaa ttccccgtga ccagactttt ggacaccagg ttggtgaatc


 


1381
agaatgcaag caggtgggaa agttttgatg tcacccccgc tgtgatgcgg tggactgcac


 


1441
agggacacgc caaccatgga ttcgtggtgg aagtggccca cttggaggag aaacaaggtg


 


1501
tctccaagag acatgttagg ataagcaggt ctttgcacca agatgaacac agctggtcac


 


1561
agataaggcc attgctagta acttttggcc atgatggaaa agggcatcct ctccacaaaa


 


1621
gagaaaaacg tcaagccaaa cacaaacagc ggaaacgcct taagtccagc tgtaagagac


 


1681
accctttgta cgtggacttc agtgacgtgg ggtggaatga ctggattgtg gctcccccgg


 


1741
ggtatcacgc cttttactgc cacggagaat gcccttttcc tctggctgat catctgaact


 


1801
ccactaatca tgccattgtt cagacgttgg tcaactctgt taactctaag attcctaagg


 


1861
catgctgtgt cccgacagaa ctcagtgcta tctcgatgct gtaccttgac gagaatgaaa


 


1921
aggttgtatt aaagaactat caggacatgg ttgtggaggg ttgtgggtgt cgctagtaca


 


1981
gcaaaattaa atacataaat atatatatat atatatattt tagaaaaaag aaaaaaacaa


 


2041
acaaacaaaa aaaccccacc ccagttgaca ctttaatatt tcccaatgaa gactttattt


 


2101
atggaatgga atggaaaaaa aaacagctat tttgaaaata tatttatatc tacgaaaaga


 


2161
agttgggaaa acaaatattt taatcagaga attattcctt aaagatttaa aatgtattta


 


2221
gttgtacatt ttatatgggt tcaaccccag cacatgaagt ataatggtca gatttatttt


 


2281
gtatttattt actattataa ccacttttta ggaaaaaaat agctaatttg tatttatatg


 


2341
taatcaaaag aagtatcggg tttgtacata attttccaaa aattgtagtt gttttcagtt


 


2401
gtgtgtattt aagatgaaaa gtctacatgg aaggttactc tggcaaagtg cttagcacgt


 


2461
ttgctttttt gcagtgctac tgttgagttc acaagttcaa gtccagaaaa aaaaagtgga


 


2521
taatccactc tgctgacttt caagattatt atattattca attctcagga atgttgcaga


 


2581
gtgattgtcc aatccatgag aatttacatc cttattaggt ggaatatttg gataagaacc


 


2641
agacattgct gatctattat agaaactctc ctcctgcccc ttaatttaca gaaagaataa


 


2701
agcaggatcc atagaaataa ttaggaaaac gatgaacctg caggaaagtg aatgatggtt


 


2761
tgttgttctt ctttcctaaa ttagtgatcc cttcaaaggg gctgatctgg ccaaagtatt


 


2821
caataaaacg taagatttct tcattattga tattgtggtc atatatattt aaaattgata


 


2881
tctcgtggcc ctcatcaagg gttggaaatt tatttgtgtt ttacctttac ctcatctgag


 


2941
agctctttat tctccaaaga acccagtttt ctaacttttt gcccaacacg cagcaaaatt


 


3001
atgcacatcg tgttttctgc ccaccctctg ttctctgacc tatcagcttg cttttctttc


 


3061
caaggttgtg tgtttgaaca catttctcca aatgttaaac ctatttcaga taataaatat


 


3121
caaatctctg gcatttcatt ctataaagtc






The amino acid sequence of human BMP-2 preproprotein, provided by Genbank Accession No. NP_001191.1, is incorporated herein by reference, and is shown below (SEQ ID NO:146).









1
mvagtrclla lllpqvllgg aaglvpelgr rkfaaassgr pssgpsdevl sefelrllsm



 


61
fglkqrptps rdavvppyml dlyrrhsgqp gspapdhrle raasrantvr sfhheeslee


 


121
lpetsgkttr rfffnlssip teefitsael qvfreqmgda lgnnssfhhr iniyeiikpa


 


181
tanskfpvtr lldtrlvnqn asrwesfdvt pavmrwtaqg hanhgfvvev ahleekqgvs


 


241
krhvrisrsl hqdehswsqi rpllvtfghd gkghplhkre krgakhkgrk rlkssckrhp


 


301
lyvdfsdvgw ndwivappgy hafychgecp fpladhlnst nhaivqtivn svnskipkac


 


361
cvptelsais mlyldenekv vlknyqdmvv egcgcr


 








(Signal protein AA 1-23; proprotein AA 24-396; mature protein AA 283-396).







The siRNA used to target human BMP-2 mRNA include following sequences (SEQ ID NO: 147-150):









SEQ NO: 147:



5′-UUGUGAACUCAACAGUAGC-3′



 



SEQ NO: 148:



5′-UUAAUUUUGCUGUACUAGC-3′



 



SEQ NO: 149:



5′-UAAAACACAAAUAAAUUUC-3′



 



SEQ NO: 150:



5′-UUCUUUCUGUAAAUUAAGG-3′






The molecular beacon used to target human BMP-2 mRNA includes the following sequences (SEQ ID NO: 151-153):









SEQ NO 151:



5′-CCGGTC TAATACAAAATAAATCTG GACCGG-3′



 



SEQ NO 152:



5′-CCGGTC AAAACACAAATAAATTTCC GACCGG-3′



 










SEQ NO 153:










5′-CCGGTC TTCATTCTCGTCAAGGTAC GACCGG-3′






The mRNA transcript sequence encoding human BMP-4 variant 1, provided by Genbank Accession No. NM_001202.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 154).









1
aagaggagga aggaagatgc gagaaggcag aggaggaggg agggagggaa ggagcgcgga



 


61
gcccggcccg gaagctaggt gagtgtggca tccgagctga gggacgcgag cctgagacgc


 


121
cgctgctgct ccggctgagt atctagcttg tctccccgat gggattcccg tccaagctat


 


181
ctcgagcctg cagcgccaca gtccccggcc ctcgcccagg ttcactgcaa ccgttcagag


 


241
gtccccagga gctgctgctg gcgagcccgc tactgcaggg acctatggag ccattccgta


 


301
gtgccatccc gagcaacgca ctgctgcagc ttccctgagc ctttccagca agtttgttca


 


361
agattggctg tcaagaatca tggactgtta ttatatgcct tgttttctgt caagacacca


 


421
tqattcctgg taaccgaatg ctgatggtcg ttttattatg ccaagtcctg ctaggaggcg


 


481
cgagccatgc tagtttgata cctgagacgg ggaagaaaaa agtcgccgag attcagggcc


 


541
acgcgggagg acgccgctca gggcagagcc atgagctcct gcgggacttc gaggcgacac


 


601
ttctgcagat gtttgggctg cgccgccgcc cgcagcctag caagagtgcc gtcattccgg


 


661
actacatgcg ggatctttac cggcttcagt ctggggagga ggaggaagag cagatccaca


 


721
gcactggtct tgagtatcct gagcgcccgg ccagccgggc caacaccgtg aggagcttcc


 


781
accacgaaga acatctggag aacatcccag ggaccagtga aaactctgct tttcgtttcc


 


841
tctttaacct cagcagcatc cctgagaacg aggtgatctc ctctgcagag cttcggctct


 


901
tccgggagca ggtggaccag ggccctgatt gggaaagggg cttccaccgt ataaacattt


 


961
atgaggttat gaagccccca gcagaagtgg tgcctgggca cctcatcaca cgactactgg


 


1021
acacgagact ggtccaccac aatgtgacac ggtgggaaac ttttgatgtg agccctgcgg


 


1081
tccttcgctg gacccgggag aagcagccaa actatgggct agccattgag gtgactcacc


 


1141
tccatcagac tcggacccac cagggccagc atgtcaggat tagccgatcg ttacctcaag


 


1201
ggagtgggaa ttgggcccag ctccggcccc tcctggtcac ctttggccat gatggccggg


 


1261
gccatgcctt gacccgacgc cggagggcca agcgtagccc taagcatcac tcacagcggg


 


1321
ccaggaagaa gaataagaac tgccggcgcc actcgctcta tgtggacttc agcgatgtgg


 


1381
gctggaatga ctggattgtg gccccaccag gctaccaggc cttctactgc catggggact


 


1441
gcccctttcc actggctgac cacctcaact caaccaacca tgccattgtg cagaccctgg


 


1501
tcaattctgt caattccagt atccccaaag cctgttgtgt gcccactgaa ctgagtgcca


 


1561
tctccatgct gtacctggat gagtatgata aggtggtact gaaaaattat caggagatgg


 


1621
tagtagaggg atgtgggtgc cgctgagatc aggcagtcct tgaggataga cagatataca


 


1681
caccacacac acacaccaca tacaccacac acacacgttc ccatccactc acccacacac


 


1741
tacacagact gcttccttat agctggactt ttatttaaaa aaaaaaaaaa aaaaggaaaa


 


1801
aatccctaaa cattcacctt gaccttattt atgactttac gtgcaaatgt tttgaccata


 


1861
ttgatcatat attttgacaa aatatattta taactacgta ttaaaagaaa aaaataaaat


 


1921
gagtcattat tttaaaggta aaaaaaaaaa aaaaaaa






The amino acid sequence of human BMP-4preproprotein, provided by Genbank Accession No. NP_001193.2, is incorporated herein by reference, and is shown below (SEQ ID NO:155).









1
mipgnrmlmv vllcqvllgg ashaslipet gkkkvaeiqg haggrrsgqs hellrdfeat



 


61
llqmfglrrr pqpsksavip dymrdlyrlq sgeeeeeqih stgleyperp asrantvrsf


 


121
hheehlenip gtsensafrf lfnlssipen evissaelrl freqvdqgpd wergfhrini


 


181
yevmkppaev vpghlitrll dtrlvhhnvt rwetfdvspa vlrwtrekqp nyglaievth


 


241
lhqtrthqgq hvrisrslpq gsgnwaqlrp llvtfghdgr ghaltrrrra krspkhhsqr


 


301
arkknkncrr hslyvdfsdv gwndwivapp gyqafychgd cpfpladhln stnhaivqtl


 


361
vnsvnssipk accvptelsa ismlyldeyd kvvlknyqem vvegcgcr


 








(Signal peptide AA 1-24)







The siRNA used to target human BMP-4 variant 1 mRNA include following sequences (SEQ ID NO: 156-159):









SEQ NO: 156:



5′-UAAUAAAACGACCAUCAGCA-3′


 



SEQ NO: 157:



5′-UAUCUGUCUAUCCUCAAGGA-3′


 



SEQ NO: 158:



5′-UUCUUAUUCUUCUUCCUGGC-3′


 



SEQ NO: 159:



5′-UAAUAAAACGACCAUCAGC-3′






The molecular beacon used to target human BMP-4 variant 1 mRNA includes the following sequences (SEQ ID NO: 160-162):









SEQ NO 160:



5′-CCGGTC TATCTGTCTATCCTCAAGG GACCGG-3′


 



SEQ NO 161:



5′-CCGGTC TCTCAGGTATCAAACTAGC GACCGG-3′


 



SEQ NO 162:



5′-CCGGTC TTTGTCAAAATATATGATC GACCGG-3′






The mRNA transcript sequence encoding human BMP-7, provided by Genbank Accession No. NM_001719.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 163).









1
agcgcgtacc actctggcgc tcccgaggcg gcctcttgtg cgatccaggg cgcacaaggc



 


61
tgggagagcg ccccggggcc cctgctatcc gcgccggagg ttggaagagg gtgggttgcc


 


121
gccgcccgag ggcgagagcg ccagaggagc gggaagaagg agcgctcgcc cgcccgcctg


 


181
cctcctcgct gcctccccgg cgttggctct ctggactcct aggcttgctg gctgctcctc


 


241
ccacccgcgc ccgcctcctc actcgccttt tcgttcgccg gggctgcttt ccaagccctg


 


301
cggtgcgccc gggcgagtgc ggggcgaggg gcccggggcc agcaccgagc agggggcggg


 


361
ggtccgggca gagcgcggcc ggccggggag gggccatgtc tggcgcgggc gcagcggggc


 


421
ccgtctgcag caagtgaccg agcggcgcgg acggccgcct gccccctctg ccacctgggg


 


481
cggtgcgggc ccggagcccg gagcccgggt agcgcgtaga gccggcgcgat gcacgtgcg


 


541
ctcactgcga gctgcggcgc cgcacagctt cgtggcgctc tgggcacccc tgttcctgct


 


601
gcgctccgcc ctggccgact tcagcctgga caacgaggtg cactcgagct tcatccaccg


 


661
gcgcctccgc agccaggagc ggcgggagat gcagcgcgag atcctctcca ttttgggctt


 


721
gccccaccgc ccgcgcccgc acctccaggg caagcacaac tcggcaccca tgttcatgct


 


781
ggacctgtac aacgccatgg cggtggagga gggcggcggg cccggcggcc agggcttctc


 


841
ctacccctac aaggccgtct tcagtaccca gggcccccct ctggccagcc tgcaagatag


 


901
ccatttcctc accgacgccg acatggtcat gagcttcgtc aacctcgtgg aacatgacaa


 


961
ggaattcttc cacccacgct accaccatcg agagttccgg tttgatcttt ccaagatccc


 


1021
agaaggggaa gctgtcacgg cagccgaatt ccggatctac aaggactaca tccgggaacg


 


1081
cttcgacaat gagacgttcc ggatcagcgt ttatcaggtg ctccaggagc acttgggcag


 


1141
ggaatcggat ctcttcctgc tcgacagccg taccctctgg gcctcggagg agggctggct


 


1201
ggtgtttgac atcacagcca ccagcaacca ctgggtggtc aatccgcggc acaacctggg


 


1261
cctgcagctc tcggtggaga cgctggatgg gcagagcatc aaccccaagt tggcgggcct


 


1321
gattgggcgg cacgggcccc agaacaagca gcccttcatg gtggctttct tcaaggccac


 


1381
ggaggtccac ttccgcagca tccggtccac ggggagcaaa cagcgcagcc agaaccgctc


 


1441
caagacgccc aagaaccagg aagccctgcg gatggccaac gtggcagaga acagcagcag


 


1501
cgaccagagg caggcctgta agaagcacga gctgtatgtc agcttccgag acctgggctg


 


1561
gcaggactgg atcatcgcgc ctgaaggcta cgccgcctac tactgtgagg gggagtgtgc


 


1621
cttccctctg aactcctaca tgaacgccac caaccacgcc atcgtgcaga cgctggtcca


 


1681
cttcatcaac ccggaaacgg tgcccaagcc ctgctgtgcg cccacgcagc tcaatgccat


 


1741
ctccgtcctc tacttcgatg acagctccaa cgtcatcctg aagaaataca gaaacatggt


 


1801
ggtccgggcc tgtggctgcc actagctcct ccgagaattc agaccctttg gggccaagtt


 


1861
tttctggatc ctccattgct cgccttggcc aggaaccagc agaccaactg ccttttgtga


 


1921
gaccttcccc tccctatccc caactttaaa ggtgtgagag tattaggaaa catgagcagc


 


1981
atatggcttt tgatcagttt ttcagtggca gcatccaatg aacaagatcc tacaagctgt


 


2041
gcaggcaaaa cctagcagga aaaaaaaaca acgcataaag aaaaatggcc gggccaggtc


 


2101
attggctggg aagtctcagc catgcacgga ctcgtttcca gaggtaatta tgagcgccta


 


2161
ccagccaggc cacccagccg tgggaggaag ggggcgtggc aaggggtggg cacattggtg


 


2221
tctgtgcgaa aggaaaattg acccggaagt tcctgtaata aatgtcacaa taaaacgaat


 


2281
gaatgaaaat ggttaggacg ttacagatat attttcctaa acaatttatc cccatttctc


 


2341
ggtttatcct gatgcgtaaa cagaagctgt gtcaagtgga gggcggggag gtccctctcc


 


2401
attccctaca gttttcatcc tgaggcttgc agaggcccag tgtttaccga ggtttgccca


 


2461
aatccaagat ctagtgggag gggaaagagc aaatgtctgc tccgaggagg gcggtgtgtt


 


2521
gatctttgga ggaaaaatat gttctgttgt tcagctggat ttgccgtggc agaaatgaaa


 


2581
ctaggtgtgt gaaatacccg cagacatttg ggattggctt ttcacctcgc cccagtggta


 


2641
gtaaatccat gtgaaattgc agaggggaca aggacagcaa gtaggatgga acttgcaact


 


2701
caaccctgtt gttaagaagc accaatgggc cgggcacagt agctcccacc tgtaatccca


 


2761
gcactttggg aggctgaggt gggcggatca tttgaggtca ggagttcgag accagcctgg


 


2821
ccaacatggt gaaaccccat ctctactaaa aatacaaaaa ttagccgggc atggtggcac


 


2881
gcacctgtaa tcccagctac tctggaggct gaggcaggag aattgcttga accccagagg


 


2941
tggaggttgc agtgagccaa gatcgtccca ctgcactcca gcttgggtga caaaacaaga


 


3001
ctccatctca aaagaaaaaa aaaacagcac caatgaagcc tagttctcca cgggagtggg


 


3061
gtgagcagga gcactgcaca tcgccccagt ggaccctctg gtctttgtct gcagtggcat


 


3121
tccaaggctg ggccctggca agggcacccg tggctgtctc ttcatttgca gaccctgatc


 


3181
agaagtctct gcaaacaaat ttgctccttg aattaagggg gagatggcat aataggaggt


 


3241
ctgatgggtg caggatgtgc tggacttaca ttgcaaatag aagccttgtt gagggtgaca


 


3301
tcctaaccaa gtgtcccgat ttggaggtgg catttctgac gtggctcttg gtgtaagcct


 


3361
gccttgcctt ggctggtgag tcccataaat agtatgcact cagcctccgg ccacaaacac


 


3421
aaggcctagg ggagggctag actgtctgca aacgttttct gcatctgtaa agaaaacaag


 


3481
gtgatcgaaa actgtggcca tgtggaaccc ggtcttgtgg gggactgttt ctccatcttg


 


3541
actcagacag ttcctggaaa caccggggct ctgtttttat tttctttgat gtttttcttc


 


3601
tttagtagct tgggctgcag cctccactct ctagtcactg gggaggagta ttttttgtta


 


3661
tgtttggttt catttgctgg cagagctggg gctttttgtg tgatccctct tggtgtgagt


 


3721
tttctgaccc aaccagcctc tggttagcat catttgtaca tttaaacctg taaatagttg


 


3781
ttacaaagca aagagattat ttatttccat ccaaagctct tttgaacacc cccccccctt


 


3841
taatccctcg ttcaggacga tgagcttgct ttccttcaac ctgtttgttt tcttatttaa


 


3901
gactatttat taatggttgg accaatgtac tcacagctgt tgcgtcgagc agtccttagt


 


3961
gaaaattctg tataaataga caaaatgaaa agggtttgac cttgcaataa aaggagacgt


 


4021
ttggttctgg caaaaaaaaa aaaaaaaaa






The amino acid sequence of human BMP-7 precursor, provided by Genbank Accession No. NP_001710.1, is incorporated herein by reference, and is shown below (SEQ ID NO:164).









1
mhvrslraaa phsfvalwap lfllrsalad fsldnevhss fihrrlrsqe rremqreils



 


61
ilglphrprp hlqgkhnsap mfmldlynam aveegggpgg qgfsypykav fstqgpplas


 


121
lqdshfltda dmvmsfvnlv ehdkeffhpr yhhrefrfdl skipegeavt aaefriykdy


 


181
irerfdnetf risvyqvlqe hlgresdlfl ldsrtlwase egwlvfdita tsnhwvvnpr


 


241
hnlglqlsve tldgqsinpk lagligrhgp qnkqpfmvaf fkatevhfrs irstgskqrs


 


301
qnrsktpknq ealrmanvae nsssdqrqac kkhelyvsfr dlgwqdwiia pegyaayyce


 


361
gecafplnsy mnatnhaivq tlvhfinpet vpkpccaptq lnaisvlyfd dssnvilkky


 


421
rnmvvracgc h


 








(signal peptide AA 1-29; mature peptide AA 293-431).







The siRNA used to target human BMP-7 mRNA include following sequences (SEQ ID NO: 165-168):









SEQ NO: 165:



5′-UUCCUAAUACUCUCACACC-3′


 



SEQ NO: 166:



5′-UAACAAAAAAUACUCCUCC-3′


 



SEQ NO: 167:



5′-UAAAUAAGAAAACAAACAGG-3′


 



SEQ NO: 168:



5′-UUCCUAAUACUCUCACACCU-3′






The molecular beacon used to target human BMP-7 mRNA includes the following sequences (SEQ ID NO: 169-171):









SEQ NO 169:



5′-CCGGTC TAACAAAAAATACTCCTCCC GACCGG-3′


 



SEQ NO 170:



5′-CCGGTC TTGTAACAACUATTTACAGG GACCGG-3′


 



SEQ NO 171:



5′-CCGGTC TAAATAAGAAAACAAACAG GACCGG-3′






The mRNA transcript sequence encoding human IL-1 receptor antagonist variant 3, provided by Genbank Accession No. NM_000577.4, is incorporated herein by reference, and is shown below (SEQ ID NO: 172).









1
gggcagctcc accctgggag ggactgtggc ccaggtactg cccgggtgct actttatggg



 


61
cagcagctca gttgagttag agtctggaag acctcagaag acctcctgtc ctatgaggcc


 


121
ctccccatgg ctttagagac gatctgccga ccctctggga gaaaatccag caagatgcaa


 


181
gccttcagaa tctgggatgt taaccagaag accttctatc tgaggaacaa ccaactagtt


 


241
gctggatact tgcaaggacc aaatgtcaat ttagaagaaa agatagatgt ggtacccatt


 


301
gagcctcatg ctctgttctt gggaatccat ggagggaaga tgtgcctgtc ctgtgtcaag


 


361
tctggtgatg agaccagact ccagctggag gcagttaaca tcactgacct gagcgagaac


 


421
agaaagcagg acaagcgctt cgccttcatc cgctcagaca gtggccccac caccagtttt


 


481
gagtctgccg cctgccccgg ttggttcctc tgcacagcga tggaagctga ccagcccgtc


 


541
agcctcacca atatgcctga cgaaggcgtc atggtcacca aattctactt ccaggaggac


 


601
gagtagtact gcccaggcct gcctgttccc attcttgcat ggcaaggact gcagggactg


 


661
ccagtccccc tgccccaggg ctcccggcta tgggggcact gaggaccagc cattgagggg


 


721
tggaccctca gaaggcgtca caacaacctg gtcacaggac tctgcctcct cttcaactga


 


781
ccagcctcca tgctgcctcc agaatggtct ttctaatgtg tgaatcagag cacagcagcc


 


841
cctgcacaaa gcccttccat gtcgcctctg cattcaggat caaaccccga ccacctgccc


 


901
aacctgctct cctcttgcca ctgcctcttc ctccctcatt ccaccttccc atgccctgga


 


961
tccatcaggc cacttgatga cccccaacca agtggctccc acaccctgtt ttacaaaaaa


 


1021
gaaaagacca gtccatgagg gaggttttta agggtttgtg gaaaatgaaa attaggattt


 


1081
catgattttt ttttttcagt ccccgtgaag gagagccctt catttggaga ttatgttctt


 


1141
tcggggagag gctgaggact taaaatattc ctgcatttgt gaaatgatgg tgaaagtaag


 


1201
tggtagcttt tcccttcttt ttcttctttt tttgtgatgt cccaacttgt aaaaattaaa


 


1261
agttatggta ctatgttagc cccataattt tttttttcct tttaaaacac ttccataatc


 


1321
tggactcctc tgtccaggca ctgctgccca gcctccaagc tccatctcca ctccagattt


 


1381
tttacagctg cctgcagtac tttacctcct atcagaagtt tctcagctcc caaggctctg


 


1441
agcaaatgtg gctcctgggg gttctttctt cctctgctga aggaataaat tgctccttga


 


1501
cattgtagag cttctggcac ttggagactt gtatgaaaga tggctgtgcc tctgcctgtc


 


1561
tcccccaccg ggctgggagc tctgcagagc aggaaacatg actcgtatat gtctcaggtc


 


1621
cctgcagggc caagcaccta gcctcgctct tggcaggtac tcagcgaatg aatgctgtat


 


1681
atgttgggtg caaagttccc tacttcctgt gacttcagct ctgttttaca ataaaatctt


 


1741
gaaaatgcct aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa


 


1801
aa






The amino acid sequence of human IL-1 receptor antagonist isoform 3, provided by Genbank Accession No. NP_000568.1, is incorporated herein by reference, and is shown below (SEQ ID NO:173).









1
maleticrps grksskmqaf riwdvnqktf ylrnnqlvag ylqgpnvnle ekidvvpiep



 


61
halflgihgg kmclscvksg detrlqleav nitdlsenrk qdkrfafirs dsgpttsfes


 


121
aacpgwflct ameadqpvsl tnmpdegvmv tkfyfqede






The Pre-miRNA sequence of human microRNA140, provided by Genbank Accession NO: NR_029681.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 174).







5′-UGUGUCUCUCUCUGUGUCCUGCCAGUGGUUUUACCCUAUGGUAGGU


 


UACGUCAUGCUGUUCUACCACAGGGUAGAACCACGGACAGGAUACCGGG


 


GCACC-3′






And mature microRNA140 (SEQ ID NO: 175).









5′-cagugguuuuacccuaugguag-3′






The Pre-miRNA sequence of human microRNA365, provided by Genbank Accession NO: NR_029854.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 176).







5′-ACCGCAGGGAAAAUGAGGGACUUUUGGGGGCAGAUGUGUUUCCAUU


 


CCACUAUCAUAAUGCCCCUAAAAAUCCUUAUUGCUCUUGCA-3′






And mature microRNA365 (SEQ ID NO: 177):









5′-AGGGACUUUUGGGGGCAGAUGUG-3′






The Pre-miRNA sequence of human microRNA125a, provided by Genbank Accession NO: NR_029693.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 178).







5′-UGCCAGUCUCUAGGUCCCUGAGACCCUUUAACCUGUGAGGACAUC


 


CAGGGUCACAGGUGAGGUUCUUGGGAGCCUGGCGUCUGGCC-3′






And two mature microRNA125a (SEQ ID NO: 179-180):









SEQ ID NO: 179: hsa-mir-125a-5p:



5′-ucccugagacccuuuaaccuguga-3′\



 



SEQ ID NO: 180: hsa-mir-125a-3p:



5′-acaggugagguucuugggagcc-3′






The mRNA sequence encoding human IL-15, provided by Genbank Accession No. BC018149.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 181).









1
actccgggtg gcaggcgccc gggggaatcc cagctgactc gctcactgcc ttcgaagtcc



 


61
ggcgcccccc gggagggaac tgggtggccg caccctcccg gctgcggtgg ctgtcgcccc


 


121
ccaccctgca gccaggactc gatggaggta cagagctcgg cttctttgcc ttgggagggg


 


181
agtggtggtg gttgaaaggg cgatggaatt ttccccgaaa gcctacgccc agggcccctc


 


241
ccagctccag cgttaccctc cggtctatcc tactggccga gctgccccgc cttctcatgg


 


301
ggaaaactta gccgcaactt caatttttgg tttttccttt aatgacactt ctgaggctct


 


361
cctagccatc ctcccgcttc cggaggagcg cagatcgcag gtccctttgc ccctggcgtg


 


421
cgactcccta ctgcgctgcg ctcttacggc gttccaggct gctggctagc gcaaggcggg


 


481
ccgggcaccc cgcgctccgc tgggagggtg agggacgcgc gtctggcggc cccagccaag


 


541
ctgcgggttt ctgagaagac gctgtcccgc agccctgagg gctgagttct gcacccagtc


 


601
aagctcagga aggccaagaa aagaatccat tccaatatat ggccatgtgg ctctttggag


 


661
caatgttcca tcatgttcca tgctgctgac gtcacatgga gcacagaaat caatgttagc


 


721
agatagccag cccatacaag atcgtattgt attgtaggag gcatcgtgga tggatggctg


 


781
ctggaaaccc cttgccatag ccagctcttc ttcaatactt aaggatttac cgtggctttg


 


841
agtaatgaga atttcgaaac cacatttgag aagtatttcc atccagtgct acttgtgttt


 


901
acttctaaac agtcattttc taactgaagc tggcattcat gtcttcattt tgggctgttt


 


961
cagtgcaggg cttcctaaaa cagaagccaa ctgggtgaat gtaataagtg atttgaaaaa


 


1021
aattgaagat cttattcaat ctatgcatat tgatgctact ttatatacgg aaagtgatgt


 


1081
tcaccccagt tgcaaagtaa cagcaatgaa gtgctttctc ttggagttac aagttatttc


 


1141
acttgagtcc ggagatgcaa gtattcatga tacagtagaa aatctgatca tcctagcaaa


 


1201
caacagtttg tcttctaatg ggaatgtaac agaatctgga tgcaaagaat gtgaggaact


 


1261
ggaggaaaaa aatattaaag aatttttgca gagttttgta catattgtcc aaatgttcat


 


1321
caacacttct tgattgcaat tgattctttt taaagtgttt ctgttattaa caaacatcac


 


1381
tctgctgctt agacataaca aaacactcgg catttcaaat gtgctgtcaa aacaagtttt


 


1441
tctgtcaaga agatgatcag accttggatc agatgaactc ttagaaatga aggcagaaaa


 


1501
atgtcattga gtaatatagt gactatgaac ttctctcaga cttactttac tcattttttt


 


1561
aatttattat tgaaattgta catatttgtg gaataatgta aaatgttgaa taaaaatatg


 


1621
tacaagtgtt gttttttaag ttgcactgat attttacctc ttattgcaaa atagcatttg


 


1681
tttaagggtg atagtcaaat tatgtattgg tggggctggg taccaatgct gcaggtcaac


 


1741
agctatgctg gtaggctcct gcctgtgtgg aaccactgac tactggctct cattgacttc


 


1801
cttactaagc atagcaaaca gaggaagaat ttgttatcag taagaaaaag aagaactata


 


1861
tgtgaatcct cttctttaca ctgtaattta gttattgatg tataaagcaa ctgttatgaa


 


1921
ataaagaaat tgcaataact ggcaaaaaaa aaaaaaaaaa aaaaaaaa






The atg start and stop codons are bolded and underlined. The amino acid sequence of human IL-15, provided by Genbank Accession No. AAH18149.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 182).








(SEQ ID NO: 182)










1
mriskphlrs isiqcylcll lnshflteag ihvfilgcfs aglpkteanw vnvisdlkki



 


61
edliqsmhid atlytesdvh psckvtamkc fllelqvisl esgdasihdt venliilann


 


121
slssngnvte sgckeceele eknikeflqs fvhivqmfin ts






The mRNA sequence encoding human IL-20 (interleukin-20 precursor), provided by Genbank Accession No. NM_018724.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 183).









1
ctttgaattc ctagctcctg tggtctccag atttcaggcc taagatgaaa gcctctagtc



 


61
ttgccttcag ccttctctct gctgcgtttt atctcctatg gactccttcc actggactga


 


121
agacactcaa tttgggaagc tgtgtgatcg ccacaaacct tcaggaaata cgaaatggat


 


181
tttctgagat acggggcagt gtgcaagcca aagatggaaa cattgacatc agaatcttaa


 


241
ggaggactga gtctttgcaa gacacaaagc ctgcgaatcg atgctgcctc ctgcgccatt


 


301
tgctaagact ctatctggac agggtattta aaaactacca gacccctgac cattatactc


 


361
tccggaagat cagcagcctc gccaattcct ttcttaccat caagaaggac ctccggctct


 


421
gtcatgccca catgacatgc cattgtgggg aggaagcaat gaagaaatac agccagattc


 


481
tgagtcactt tgaaaagctg gaacctcagg cagcagttgt gaaggctttg ggggaactag


 


541
acattcttct gcaatggatg gaggagacag aataggagga aagtgatgct gctgctaaga


 


601
atattcgagg tcaagagctc cagtcttcaa tacctgcaga ggaggcatga ccccaaacca


 


661
ccatctcttt actgtactag tcttgtgctg gtcacagtgt atcttattta tgcattactt


 


721
gcttccttgc atgattgtct ttatgcatcc ccaatcttaa ttgagaccat acttgtataa


 


781
gatttttgta atatctttct gctattggat atatttatta gttaatatat ttatttattt


 


841
tttgctattt aatgtattta tttttttact tggacatgaa actttaaaaa aattcacaga


 


901
ttatatttat aacctgacta gagcaggtga tgtattttta tacagtaaaa aaaaaaaacc


 


961
ttgtaaattc tagaagagtg gctagggggg ttattcattt gtattcaact aaggacatat


 


1021
ttactcatgc tgatgctctg tgagatattt gaaattgaac caatgactac ttaggatggg


 


1081
ttgtggaata agttttgatg tggaattgca catctacctt acaattactg accatcccca


 


1141
gtagactccc cagtcccata attgtgtatc ttccagccag gaatcctaca cggccagcat


 


1201
gtatttctac aaataaagtt ttctttgcat aacaaaaaaa aaaaaaaaaa aa






The atg start and stop codons are bolded and underlined. The amino acid sequence of human IL-20 (interleukin-20 precursor), provided by Genbank Accession No. NP_061194.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 184).









1
mkasslafsl lsaafyllwt pstglktlnl gscviatnlq eirngfseir gsvqakdgni



 


61
dirilrrtes lqdtkpanrc cllrhllrly ldrvfknyqt pdhytlrkis slansfltik


 


121
kdlrlchahm tchcgeeamk kysqilshfe klepqaavvk algeldillq wmeete






The mRNA sequence encoding human PADI4 (protein-arginine deiminase type-4), provided by Genbank Accession No. NM_012387.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 185).








(SEQ ID NO: 185)










1
acagccagag ggacgagcta gcccgacgat ggcccagggg acattgatcc gtgtgacccc



 


61
agagcagccc acccatgccg tgtgtgtgct gggcaccttg actcagcttg acatctgcag


 


121
ctctgcccct gaggactgca cgtccttcag catcaacgcc tccccagggg tggtcgtgga


 


181
tattgcccac ggccctccag ccaagaagaa atccacaggt tcctccacat ggcccctgga


 


241
ccctggggta gaggtgaccc tgacgatgaa agtggccagt ggtagcacag gcgaccagaa


 


301
ggttcagatt tcatactacg gacccaagac tccaccagtc aaagctctac tctacctcac


 


361
cggggtggaa atctccttgt gcgcagacat cacccgcacc ggcaaagtga agccaaccag


 


421
agctgtgaaa gatcagagga cctggacctg gggcccttgt ggacagggtg ccatcctgct


 


481
ggtgaactgt gacagagaca atctcgaatc ttctgccatg gactgcgagg atgatgaagt


 


541
gcttgacagc gaagacctgc aggacatgtc gctgatgacc ctgagcacga agacccccaa


 


601
ggacttcttc acaaaccata cactggtgct ccacgtggcc aggtctgaga tggacaaagt


 


661
gagggtgttt caggccacac ggggcaaact gtcctccaag tgcagcgtag tcttgggtcc


 


721
caagtggccc tctcactacc tgatggtccc cggtggaaag cacaacatgg acttctacgt


 


781
ggaggccctc gctttcccgg acaccgactt cccggggctc attaccctca ccatctccct


 


841
gctggacacg tccaacctgg agctccccga ggctgtggtg ttccaagaca gcgtggtctt


 


901
ccgcgtggcg ccctggatca tgacccccaa cacccagccc ccgcaggagg tgtacgcgtg


 


961
cagtattttt gaaaatgagg acttcctgaa gtcagtgact actctggcca tgaaagccaa


 


1021
gtgcaagctg accatctgcc ctgaggagga gaacatggat gaccagtgga tgcaggatga


 


1081
aatggagatc ggctacatcc aagccccaca caaaacgctg cccgtggtct tcgactctcc


 


1141
aaggaacaga ggcctgaagg agtttcccat caaacgcgtg atgggtccag attttggcta


 


1201
tgtaactcga gggccccaaa cagggggtat cagtggactg gactcctttg ggaacctgga


 


1261
agtgagcccc ccagtcacag tcaggggcaa ggaatacccg ctgggcagga ttctcttcgg


 


1321
ggacagctgt tatcccagca atgacagccg gcagatgcac caggccctgc aggacttcct


 


1381
cagtgcccag caggtgcagg cccctgtgaa gctctattct gactggctgt ccgtgggcca


 


1441
cgtggacgag ttcctgagct ttgtgccagc acccgacagg aagggcttcc ggctgctcct


 


1501
ggccagcccc aggtcctgct acaaactgtt ccaggagcag cagaatgagg gccacgggga


 


1561
ggccctgctg ttcgaaggga tcaagaaaaa aaaacagcag aaaataaaga acattctgtc


 


1621
aaacaagaca ttgagagaac ataattcatt tgtggagaga tgcatcgact ggaaccgcga


 


1681
gctgctgaag cgggagctgg gcctggccga gagtgacatc attgacatcc cgcagctctt


 


1741
caagctcaaa gagttctcta aggcggaagc ttttttcccc aacatggtga acatgctggt


 


1801
gctagggaag cacctgggca tccccaagcc cttcgggccc gtcatcaacg gccgctgctg


 


1861
cctggaggag aaggtgtgtt ccctgctgga gccactgggc ctccagtgca ccttcatcaa


 


1921
cgacttcttc acctaccaca tcaggcatgg ggaggtgcac tgcggcacca acgtgcgcag


 


1981
aaagcccttc tccttcaagt ggtggaacat ggtgccctga gcccatcttc cctggcgtcc


 


2041
tctccctcct ggccagatgt cgctgggtcc tctgcagtgt ggcaagcaag agctcttgtg


 


2101
aatattgtgg ctccctgggg gcggccagcc ctcccagcag tggcttgctt tcttctcctg


 


2161
tgatgtccca gtttcccact ctgaagatcc caacatggtc ctagcactgc acactcagtt


 


2221
ctgctctaag aagctgcaat aaagtttttt taagtcactt tgtac






The atg start and stop codons are bolded and underlined. The amino acid sequence of human PAD14 (protein-arginine deiminase type-4) provided by Genbank Accession No. NP_036519.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 186).








(SEQ ID NO: 186)










1
maqgtlirvt peqpthavcv lgtltqldic ssapedctsf sinaspgvvv diahgppakk



 


61
kstgsstwpl dpgvevtltm kvasgstgdq kvqisyygpk tppvkallyl tgveislcad


 


121
itrtgkvkpt ravkdqrtwt wgpcgqgail lvncdrdnle ssamdcedde vldsedlqdm


 


181
slmtlstktp kdfftnhtlv lhvarsemdk vrvfqatrgk lsskcsvvlg pkwpshylmv


 


241
pggkhnmdfy vealafpdtd fpglitltis lldtsnlelp eavvfqdsvv frvapwimtp


 


301
ntqppqevya csifenedfl ksvttlamka kcklticpee enmddqwmqd emeigyiqap


 


361
hktlpvvfds prnrglkefp ikrvmgpdfg yvtrgpqtgg isgldsfgnl evsppvtvrg


 


421
keyplgrilf gdscypsnds rqmhqalqdf lsaqqvqapv klysdwlsvg hvdeflsfvp


 


481
apdrkgfrll lasprscykl fqeqqneghg eallfegikk kkqqkiknil snktlrehns


 


541
fvercidwnr ellkrelgla esdiidipql fklkefskae affpnmvnml vlgkhlgipk


 


601
pfgpvingrc cleekvcsll eplglqctfi ndfftyhirh gevhcgtnvr rkpfsfkwwn


 


661
mvp






The mRNA sequence encoding human HLA-DRB1, provided by Genbank Accession No. HQ267233.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 187).









1
atggtgtgtc tgaggctccc tggaggctcc tgcatggcag ttctgacagt gacactgatg



 


61
gtgctgagct ccccactggc tttggctggg gacaccagac cacgtttctt ggaggaggtt


 


121
aagtttgagt gtcatttctt caacgggacg gagcgggtgc ggttgctgga aagacgcgtc


 


181
cataaccaag aggagtacgc gcgctacgac agcgacgtgg gggagtaccg ggcggtgacg


 


241
gagctggggc ggcctgatgc cgagtactgg aacagccaga aggacctcct ggagcggagg


 


301
cgtgccgcgg tggacaccta ctgcagacac aactacgggg ttggtgagag cttcacagtg


 


361
cagcggcgag ttcaacctaa ggtgactgtg tatccttcaa agacccagcc cctgcagcac


 


421
cacaacctcc tggtctgttc tgtgaatggt ttctatccag gcagcattga agtcaggtgg


 


481
ttccggaacg gccaggaaga gaagactggg gtggtgtcca cgggcctgat ccagaatgga


 


541
gactggacct tccagaccct ggtgatgctg gaaacagttc ctcagagtgg agaggtttac


 


601
acctgccaag tggagcaccc aagtgtgatg agccctctca cagtggaatg gagagcacgg


 


661
tctgaatctg cacagagcaa gatgctgagt ggagtcgggg gctttgtgct gggcctgctc


 


721
ttccttgggg ccgggctgtt catctacttc aggaatcaga aaggacactc tggacttccg


 


781
ccaacaggat tcctgagctg a






The atg start and stop codons are bolded and underlined. The amino acid sequence of human HLA-DRB1, provided by Genbank Accession No. ADZ73424.1, is incorporated herein be reference, and is shown below (SEQ ID NO: 188).








(SEQ ID NO: 188)










1
mvclrlpggs cmavltvtlm vlssplalag dtrprfleev kfechffngt ervrllerrv



 


61
hnqeeyaryd sdvgeyravt elgrpdaeyw nsqkdllerr raavdtycrh nygvgesftv


 


121
qrrvqpkvtv ypsktqplqh hnllvcsvng fypgsievrw frngqeektg vvstgliqng


 


181
dwtfqtlvml etvpqsgevy tcqvehpsvm spltvewrar sesaqskmls gvggfvlgll


 


241
flgaglfiyf rnqkghsglp ptgfls






The mRNA sequence encoding human PTPN22 provided by Genbank Accession No. BC071670.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 189).









1
ggtgtctcgg ccatgacaca catttgacat gccctccctc aacctactta tagactattt



 


61
ttcttgctct gcagcatgga ccaaagagaa attctgcaga agttcctgga tgaggcccaa


 


121
agcaagaaaa ttactaaaga ggagtttgcc aatgaatttc tgaagctgaa aaggcaatct


 


181
accaagtaca aggcagacaa aacctatcct acaactgtgg ctgagaagcc caagaatatc


 


241
aagaaaaaca gatataagga tattttgccc tatgattata gccgggtaga actatccctg


 


301
ataacctctg atgaggattc cagctacatc aatgccaact tcattaaggg agtttatgga


 


361
cccaaggctt atattgccac ccagggtcct ttatctacaa ccctcctgga cttctggagg


 


421
atgatttggg aatatagtgt ccttatcatt gttatggcat gcatggagta tgaaatggga


 


481
aagaaaaagt gtgagcgcta ctgggctgag ccaggagaga tgcagctgga atttggccct


 


541
ttctctgtat cctgtgaagc tgaaaaaagg aaatctgatt atataatcag gactctaaaa


 


601
gttaagttca atagtgaaac tcgaactatc taccagtttc attacaagaa ttggccagac


 


661
catgatgtac cttcatctat agaccctatt cttgagctca tctgggatgt acgttgttac


 


721
caagaggatg acagtgttcc catatgcatt cactgcagtg ctggctgtgg aaggactggt


 


781
gttatttgtg ctattgatta tacatggatg ttgctaaaag atgggagtca agcaaagcat


 


841
tgtattcctg agaaaaatca cactctccaa gcagactctt attctcctaa tttaccaaaa


 


901
agtaccacaa aagcagcaaa aatgatgaac caacaaagga caaaaatgga aatcaaagaa


 


961
tcttcttcct ttgactttag gacttctgaa ataagtgcaa aagaagagct agttttgcac


 


1021
cctgctaaat caagcacttc ttttgacttt ctggagctaa attacagttt tgacaaaaat


 


1081
gctgacacaa ccatgaaatg gcagacaaag gcatttccaa tagttgggga gcctcttcag


 


1141
aagcatcaaa gtttggattt gggctctctt ttgtttgagg gatgttctaa ttctaaacct


 


1201
gtaaatgcag caggaagata ttttaattca aaggtgccaa taacacggac caaatcaact


 


1261
ccttttgaat tgatacagca gagagaaacc aaggaggtgg acagcaagga aaacttttct


 


1321
tatttggaat ctcaaccaca tgattcttgt tttgtagaga tgcaggctca aaaagtaatg


 


1381
catgtttctt cagcagaact gaattattca ctgccatatg actctaaaca ccaaatacgt


 


1441
aatgcctcta atgtaaagca ccatgactct agtgctcttg gtgtatattc ttacatacct


 


1501
ttagtggaaa atccttattt ttcatcatgg cctccaagtg gtaccagttc taagatgtct


 


1561
cttgatttac ctgagaagca agatggaact gtttttcctt cttctctgtt gccaacatcc


 


1621
tctacatccc tcttctctta ttacaattca catgattctt tatcactgaa ttctccaacc


 


1681
aatatttcct cactattgaa ccaggagtca gctgtactag caactgctcc aaggatagat


 


1741
gatgaaatcc cccctccact tcctgtacgg acacctgaat catttattgt ggttgaggaa


 


1801
gctggagaat tctcaccaaa tgttcccaaa tccttatcct cagctgtgaa ggtaaaaatt


 


1861
ggaacatcac tggaatgggg tggaacatct gaaccaaaga aatttgatga ctctgtgata


 


1921
cttagaccaa gcaagagtgt aaaactccga agtcctaaat cagaactaca tcaagatcgt


 


1981
tcttctcccc cacctcctct cccagaaaga actctagagt ccttctttct tgccgatgaa


 


2041
gattgtatgc aggcccaatc tatagaaaca tattctacta gctatcctga caccatggaa


 


2101
aattcaacat cttcaaaaca gacactgaag actcctggaa aaagtttcac aaggagtaag


 


2161
agtttgaaaa ttttgcgaaa catgaaaaag agtatctgta attcttgccc accaaacaag


 


2221
cctgcagaat ctgttcagtc aaataactcc agctcatttc tgaattttgg ttttgcaaac


 


2281
cgtttttcaa aacccaaagg accaaggaat ccaccaccaa cttggaatat ttaataaaac


 


2341
tccagattta taataatatg ggctgcaagt acacctgcaa ataaaactac tagaatactg


 


2401
ctagttaaaa taagtgctct atatgcataa tatcaaatat gaagatatgc taatgtgtta


 


2461
atagctttta aaagaaaagc aaaatgccaa taagtgccag ttttgcattt tcatatcatt


 


2521
tgcattgagt tgaaaactgc aaataaaagt ttgtcacttg agcttatgta cagaatgcta


 


2581
tatgagaaac acttttagaa tggatttatt tttcattttt gccagttatt tttattttct


 


2641
tttacttttt tacataaaca taaacttcaa aaggtttgta agatttggat ctcaactaat


 


2701
ttctacattg ccagaatata ctataaaaag ttaaaaaaaa aacttacttt gtgggttgca


 


2761
atacaaactg ctcttgacaa tgactattcc ctgacagtta tttttgccta aatggagtat


 


2821
accttgtaaa tcttcccaaa tgttgtggaa aactggaata ttaagaaaat gagaaattat


 


2881
atttattaga ataaaatgtg caaataatga caattatttg aatgtaacaa ggaattcaac


 


2941
tgaaatcctg ataagtttta accaaagtca ttaaattacc aattctagaa aagtaatcaa


 


3001
tgaaatataa tagctatctt ttggtagcaa aagatataaa ttgtatatgt ttatacagga


 


3061
tctttcagat catgtgcaat ttttatctaa ccaatcagaa atactagttt aaaatgaatt


 


3121
tctatatgaa tatggatctg ccataagaaa atctagttca actctaattt tatgtagtaa


 


3181
ataaattggc aggtaattgt ttttacaaag aatccacctg acttccccta atgcattaaa


 


3241
aatattttta tttaaataac tttatttata acttttagaa acatgtagta ttgtttaaac


 


3301
atcatttgtt cttcagtatt tttcatttgg aagtccaata gggcaaattg aatgaagtat


 


3361
tattatctgt ctcttgtagt acaatgtatc caacagacac tcaataaact ttttggttgt


 


3421
taaaaaaaaa aaaaaa






The atg start and stop codons are bolded and underlined. The amino acid sequence of human PTPN22, provided by Genbank Accession No. AAH716701.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 190).








(SEQ ID NO: 190)










1
mdqreilqkf ldeaqskkit keefaneflk lkrqstkyka dktypttvae kpknikknry



 


61
kdilpydysr velslitsde dssyinanfi kgvygpkayi atqgplsttl ldfwrmiwey


 


121
svliivmacm eyemgkkkce rywaepgemq lefgpfsvsc eaekrksdyi irtlkvkfns


 


181
etrtiyqfhy knwpdhdvps sidpileliw dvrcyqedds vpicihcsag cgrtgvicai


 


241
dytwmllkdg sqakhcipek nhtlqadsys pnlpksttka akmmnqqrtk meikesssfd


 


301
frtseisake elvlhpakss tsfdflelny sfdknadttm kwqtkafpiv geplqkhqsl


 


361
dlgsllfegc snskpvnaag ryfnskvpit rtkstpfeli qqretkevds kenfsylesq


 


421
phdscfvemq aqkvmhvssa elnyslpyds khqirnasnv khhdssalgv ysyiplvenp


 


481
yfsswppsgt sskmsldlpe kqdgtvfpss llptsstslf syynshdsls lnsptnissl


 


541
lnqesavlat apriddeipp plpvrtpesf ivveeagefs pnvpkslssa vkvkigtsle


 


601
wggtsepkkf ddsvilrpsk svklrspkse lhqdrssppp plpertlesf fladedcmqa


 


661
qsietystsy pdtmenstss kqtlktpgks ftrskslkil rnmkksicns cppnkpaesv


 


721
qsnnsssfln fgfanrfskp kgprnppptw ni






The mRNA sequence encoding human TNFAIP3 provided by Genbank Accession No. BC114480.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 191).









1
ccggagaggt gttggagagc acaatggctg aacaagtcct tcctcaggct ttgtatttga



 


61
gcaatatgcg gaaagctgtg aagatacggg agagaactcc agaagacatt tttaaaccta


 


121
ctaatgggat cattcatcat tttaaaacca tgcaccgata cacactggaa atgttcagaa


 


181
cttgccagtt ttgtcctcag tttcgggaga tcatccacaa agccctcatc gacagaaaca


 


241
tccaggccac cctggaaagc cagaagaaac tcaactggtg tcgagaagtc cggaagcttg


 


301
tggcgctgaa aacgaacggt gacggcaatt gcctcatgca tgccacttct cagtacatgt


 


361
ggggcgttca ggacacagac ttggtactga ggaaggcgct gttcagcacg ctcaaggaaa


 


421
cagacacacg caactttaaa ttccgctggc aactggagtc tctcaaatct caggaatttg


 


481
ttgaaacggg gctttgctat gatactcgga actggaatga tgaatgggac aatcttatca


 


541
aaatggcttc cacagacaca cccatggccc gaagtggact tcagtacaac tcactggaag


 


601
aaatacacat atttgtcctt tgcaacatcc tcagaaggcc aatcattgtc atttcagaca


 


661
aaatgctaag aagtttggaa tcaggttcca atttcgcccc tttgaaagtg ggtggaattt


 


721
acttgcctct ccactggcct gcccaggaat gctacagata ccccattgtt ctcggctatg


 


781
acagccatca ttttgtaccc ttggtgaccc tgaaggacag tgggcctgaa atccgagctg


 


841
ttccacttgt taacagagac cggggaagat ttgaagactt aaaagttcac tttttgacag


 


901
atcctgaaaa tgagatgaag gagaagctct taaaagagta cttaatggtg atagaaatcc


 


961
ccgtccaagg ctgggaccat ggcacaactc atctcatcaa tgccgcaaag ttggatgaag


 


1021
ctaacttacc aaaagaaatc aatctggtag atgattactt tgaacttgtt cagcatgagt


 


1081
acaagaaatg gcaggaaaac agcgagcagg ggaggagaga ggggcacgcc cagaatccca


 


1141
tggaaccttc cgtgccccag ctttctctca tggatgtaaa atgtgaaacg cccaactgcc


 


1201
ccttcttcat gtctgtgaac acccagcctt tatgccatga gtgctcagag aggcggcaaa


 


1261
agaatcaaaa caaactccca aagctgaact ccaagccggg ccctgagggg ctccctggca


 


1321
tggcgctcgg ggcctctcgg ggagaagcct atgagccctt ggcgtggaac cctgaggagt


 


1381
ccactggggg gcctcattcg gccccaccga cagcacccag cccttttctg ttcagtgaga


 


1441
ccactgccat gaagtgcagg agccccggct gccccttcac actgaatgtg cagcacaacg


 


1501
gattttgtga acgttgccac aacgcccggc aacttcacgc cagccacgcc ccagaccaca


 


1561
caaggcactt ggatcccggg aagtgccaag cctgcctcca ggatgttacc aggacattta


 


1621
atgggatctg cagtacttgc ttcaaaagga ctacagcaga ggcctcctcc agcctcagca


 


1681
ccagcctccc tccttcctgt caccagcgtt ccaagtcaga tccctcgcgg ctcgtccgga


 


1741
gcccctcccc gcattcttgc cacagagctg gaaacgacgc ccctgctggc tgcctgtctc


 


1801
aagctgcacg gactcctggg gacaggacgg ggacgagcaa gtgcagaaaa gccggctgcg


 


1861
tgtattttgg gactccagaa aacaagggct tttgcacact gtgtttcatc gagtacagag


 


1921
aaaacaaaca ttttgctgct gcctcaggga aagtcagtcc cacagcgtcc aggttccaga


 


1981
acaccattcc gtgcctgggg agggaatgcg gcacccttgg aagcaccatg tttgaaggat


 


2041
actgccagaa gtgtttcatt gaagctcaga atcagagatt tcatgaggcc aaaaggacag


 


2101
aagagcaact gagatcgagc cagcgcagag atgtgcctcg aaccacacaa agcacctcaa


 


2161
ggcccaagtg cgcccgggcc tcctgcaaga acatcctggc ctgccgcagc gaggagctct


 


2221
gcatggagtg tcagcatccc aaccagagga tgggccctgg ggcccaccgg ggtgagcctg


 


2281
cccccgaaga cccccccaag cagcgttgcc gggcccccgc ctgtgatcat tttggcaatg


 


2341
ccaagtgcaa cggctactgc aacgaatgct ttcagttcaa gcagatgtat ggctaaccgg


 


2401
aaacaggtgg gtcacctcct gcaagaagtg gggcctcgag ctgtcagtca tcatggtgct


 


2461
atcctctgaa cccctcagct gccactgcaa cagtgggctt aagggtgtct gagcaggaga


 


2521
ggaaagataa gctcttcgtg gtgcccacga tgctcaggtt tggtaacccg ggagtgttcc


 


2581
caggtggcct tagaaagcaa agcttgtaac tggcaaggga tgatgtcaga ttcagcccaa


 


2641
ggttcctcct ctcctaccaa gcaggaggcc aggaacttct ttggacttgg aaggtgtgcg


 


2701
gggactggcc gaggcccctg caccctgcgc atcaggactg cttcatcgtc ttggctgaga


 


2761
aagggaaaag acacacaagt cgcgtgggtt ggagaagcca gagccattcc acctcccctc


 


2821
ccccagcatc tctcagagat gtgaagccag atcctcatgg cagcgaggcc ctctgcaaga


 


2881
agctcaagga agctcaggga aaatggacgt attcagagag tgtttgtagt tcatggtttt


 


2941
tccctacctg cccggttcct ttcctgagga cccggcagaa atgcagaacc atccatggac


 


3001
tgtgattctg aggctgctga gactgaacat gttcacattg acagaaaaac aagctgctct


 


3061
ttataatatg caccttttaa aaaattagaa tattttactg ggaagacgtg taactctttg


 


3121
ggttattact gtctttactt ctaaagaagt tagcttgaac tgaggagtaa aagtgtgtac


 


3181
atatataata tacccttaca ttatgtatga gggatttttt taaattatat tgaaatgctg


 


3241
ccctagaagt acaataggaa ggctaaataa taataacctg ttttctggtt gttgttgggg


 


3301
catgagcttg tgtatacact gcttgcataa actcaaccag ctgccttttt aaagggagct


 


3361
ctagtccttt ttgtgtaatt cactttattt attttattac aaacttcaag attatttaag


 


3421
cgaagatatt tcttcagctc tggggaaaat gccacagtgt tctcctgaga gaacatcctt


 


3481
gctttgagtc aggctgtggg caagttcctg accacaggga gtaaattggc ctctttgata


 


3541
cacttttgct tgcctcccca ggaaagaagg aattgcatcc aaggtataca tacatattca


 


3601
tcgatgtttc gtgcttctcc ttatgaaact ccagctatgt aataaaaaac tatactctgt


 


3661
gttctgttaa tgcctctgag tgtcctacct ccttggagat gagataggga aggagcaggg


 


3721
atgagactgg caatggtcac agggaaagat gtggcctttt gtgatggttt tattttctgt


 


3781
taacactgtg tcctgggggg gctgggaagt cccctgcatc ccatg






The atg start and stop codons are bolded and underlined. The amino acid sequence of human TNFAIP3, provided by Genbank Accession No. AAI14481.1, is incorporated herein be reference, and is shown below (SEQ ID NO: 192).









1
maeqvlpqal ylsnmrkavk irertpedif kptngiihhf ktmhrytlem frtcqfcpqf



 


61
reiihkalid rniqatlesq kklnwcrevr klvalktngd gnclmhatsq ymwgvqdtdl


 


121
vlrkalfstl ketdtrnfkf rwqleslksq efvetglcyd trnwndewdn likmastdtp


 


181
marsglqyns leeihifvlc nilrrpiivi sdkmlrsles gsnfaplkvg giylplhwpa


 


241
qecyrypivl gydshhfvpl vtlkdsgpei ravplvnrdr grfedlkvhf ltdpenemke


 


301
kllkeylmvi eipvqgwdhg tthlinaakl deanlpkein lvddyfelvq heykkwqens


 


361
eqgrreghaq npmepsvpql slmdvkcetp ncpffmsvnt qplchecser rqknqnklpk


 


421
lnskpgpegl pgmalgasrg eayeplawnp eestggphsa pptapspflf settamkcrs


 


481
pgcpftlnvq hngfcerchn arqlhashap dhtrhldpgk cqaclqdvtr tfngicstcf


 


541
krttaeasss lstslppsch qrsksdpsrl vrspsphsch ragndapagc lsqaartpgd


 


601
rtgtskcrka gcvyfgtpen kgfctlcfie yrenkhfaaa sgkvsptasr fqntipclgr


 


661
ecgtlgstmf egycqkcfie aqnqrfheak rteeqlrssq rrdvprttqs tsrpkcaras


 


721
cknilacrse elcmecqhpn qrmgpgahrg epapedppkq rcrapacdhf gnakcngycn


 


781
ecfqfkqmyg






The mRNA sequence encoding human STAT4 provided by Genbank Accession No. L78440.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 193).









1
gctttctcct agggactgtg aggggcgctt ctgactttgg acttgagcac tgcctgggac



 


61
ctgtgctgag agagcgctag catgtctcag tggaatcaag tccaacagtt agaaatcaag


 


121
tttttggagc aggtggatca attctatgat gacaactttc ccatggaaat tcggcatctg


 


181
ttggcccaat ggattgaaaa tcaagactgg gaggcagctt ctaacaatga aaccatggca


 


241
acgattcttc ttcaaaactt gttaatacaa ctggatgaac agttaggtcg tgtttccaaa


 


301
gagaaaaacc tactcttgat acacaatcta aaaagaatta ggaaggtcct tcagggaaaa


 


361
tttcatggaa atccaatgca tgtagctgtg gttatttcaa actgtttaag ggaagagagg


 


421
agaatattgg ctgcagccaa catgcctgtc caggggcctc tagagaaatc cttacaaagt


 


481
tcttcagttt cagaaagaca gaggaatgtg gagcacaaag tggctgccat taaaaacagt


 


541
gtgcagatga cagaacaaga taccaaatac ttagaagatc tgcaagacga atttgactac


 


601
aggtataaaa caattcagac aatggatcag agtgacaaga atagtgccat ggtgaatcag


 


661
gaagttttga cactgcagga aatgcttaac agcctcgatt tcaagagaaa ggaggctctc


 


721
agtaaaatga cccaaatcat ccatgagaca gacctgttaa tgaacaccat gctcatagaa


 


781
gagctgcaag actggaagcg gcggcagcaa atcgcctgca tcgggggtcc actccacaat


 


841
gggctcgacc agcttcagaa ctgctttaca ctattggcag aaagtctttt ccaactgaga


 


901
aggcaattgg agaaactaga ggagcaatct accaaaatga catatgaagg tgatcccatt


 


961
ccaatgcaaa gaactcacat gctagaaaga gtcaccttct tgatctacaa ccttttcaag


 


1021
aactcatttg tggttgagcg acagccatgt atgccaaccc accctcagag gccgttggta


 


1081
cttaaaaccc taattcagtt cactgtaaaa ctaaggctac taataaaatt gccagaacta


 


1141
aactatcagg taaaggttaa ggcatcaatt gacaagaatg tttcaactct aagcaaccga


 


1201
agatttgtac tttgtggaac taatgtcaaa gccatgtcta ttgaagaatc ttccaatggg


 


1261
agtctctcag tagaatttcg acatttgcaa ccaaaggaaa tgaagtccag tgctggaggt


 


1321
aaaggaaatg agggctgtca catggtgact gaagaacttc attccataac gtttgaaaca


 


1381
cagatctgcc tctatggcct gaccatagat ttggagacca gctcattgcc tgtggtgatg


 


1441
atttccaatg tcagtcagtt acctaatgct tgggcatcca tcatttggta caacgtgtca


 


1501
accaacgatt cccagaactt ggttttcttt aataatcctc cacctgccac attgagtcaa


 


1561
ctactggagg tgatgagctg gcagttttca tcgtacgttg gtcgtggtct taactcagat


 


1621
caactccata tgctggcaga gaagcttaca gtccaatcta gctacagtga tggtcacctc


 


1681
acctgggcca agttctgcaa ggaacattta cctggtaaat catttacctt ttggacatgg


 


1741
cttgaagcaa tattggatct aattaagaaa cacattcttc ccctttggat tgatgggtat


 


1801
gtcatgggct ttgttagcaa agagaaggaa cggctgttgc taaaggataa aatgcctggc


 


1861
acctttttat taagattcag tgaaagccat ctcggaggaa taactttcac ctgggtggac


 


1921
cattctgaaa gtggggaagt gagattccac tctgtagaac cctacaataa aggccggttg


 


1981
tctgctctgc cattcgctga catcctgcga gactacaaag ttattatggc tgaaaacatt


 


2041
cctgaaaacc ctctgaagta cctatatcct gacattccca aagacaaagc cttcggtaaa


 


2101
cactacagct ctcagccttg cgaagtttca agaccaacag aaaggggtga caaaggttat


 


2161
gttccttctg tttttatccc catctcaaca atccgaagtg attcaacaga gccacattct


 


2221
ccatcagacc ttcttcccat gtctccaagt gtgtatgcgg tgttgagaga aaacctgagt


 


2281
cccacaacaa ttgaaactgc aatgaagtct ccttattctg ctgaatgaca ggataaactc


 


2341
tgacgcacca agaaaggaag caaatgaaaa agtttaaaga ctgttctttg cccaataacc


 


2401
acattttatt tcttcagctt tgtaaatacc aggttctagg aaatgtttga catctgaagc


 


2461
tctcttcaca ctcccgtggc actcctcaat tgggagtgtt gtgactgaaa tgcttgaaac


 


2521
caaagcttca gataaacttg caagataaga caactttaag aaaccagtgt taataacaat


 


2581
attaacag






The atg start and stop codons are bolded and underlined. The amino acid sequence of human STAT4, provided by Genbank Accession No. AAB05605.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 194).









1
msqwnqvqql eikfleqvdq fyddnfpmei rhllaqwien qdweaasnne tmatillqnl



 


61
liqldeqlgr vskeknllli hnlkrirkvl qgkfhgnpmh vavvisnclr eerrilaaan


 


121
mpvqgpleks lqsssvserq rnvehkvaai knsvqmteqd tkyledlqde fdyryktiqt


 


181
mdqsdknsam vnqevltlqe mlnsldfkrk ealskmtqii hetdllmntm lieelqdwkr


 


241
rqqiaciggp lhngldqlqn cftllaeslf qlrrqlekle eqstkmtyeg dpipmqrthm


 


301
lervtfliyn lfknsfvver qpcmpthpqr plvlktliqf tvklrllikl pelnyqvkvk


 


361
asidknvstl snrrfvlcgt nvkamsiees sngslsvefr hlqpkemkss aggkgnegch


 


421
mvteelhsit fetqiclygl tidletsslp vvmisnvsql pnawasiiwy nvstndsqnl


 


481
vffnnpppat lsqllevmsw qfssyvgrgl nsdqlhmlae kltvqssysd ghltwakfck


 


541
ehlpgksftf wtwleaildl ikkhilplwi dgyvmgfvsk ekerlllkdk mpgtfllrfs


 


601
eshlggitft wvdhsesgev rfhsvepynk grlsalpfad ilrdykvima enipenplky


 


661
lypdipkdka fgkhyssqpc evsrptergd kgyvpsvfip istirsdste phspsdllpm


 


721
spsvyavlre nlspttieta mkspysae






The mRNA sequence encoding human CCR6 provided by Genbank Accession No. AY242126.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 195).









1
atgagcgggg aatcaatgaa tttcagcgat gttttcgact ccagtgaaga ttattttgtg



 


61
tcagtcaata cttcatatta ctcagttgat tctgagatgt tactgtgctc cttgcaggag


 


121
gtcaggcagt tctccaggct atttgtaccg attgcctact ccttgatctg tgtctttggc


 


181
ctcctgggga atattctggt ggtgatcacc tttgcttttt ataagaaggc caggtctatg


 


241
acagacgtct atctcttgaa catggccatt gcagacatcc tctttgttct tactctccca


 


301
ttctgggcag tgagtcatgc cactggtgcg tgggttttca gcaatgccac gtgcaagttg


 


361
ctaaaaggca tctatgccat caactttaac tgcgggatgc tgctcctgac ttgcattagc


 


421
atggaccggt acatcgccat tgtacaggcg actaagtcat tccggctccg atccagaaca


 


481
ctaccgcgca gcaaaatcat ctgccttgtt gtgtgggggc tgtcagtcat catctccagc


 


541
tcaacttttg tcttcaacca aaaatacaac acccaaggca gcgatgtctg tgaacccaag


 


601
taccagactg tctcggagcc catcaggtgg aagctgctga tgttggggct tgagctactc


 


661
tttggtttct ttatcccttt gatgttcatg atattttgtt acacgttcat tgtcaaaacc


 


721
ttggtgcaag ctcagaattc taaaaggcac aaagccatcc gtgtaatcat agctgtggtg


 


781
cttgtgtttc tggcttgtca gattcctcat aacatggtcc tgcttgtgac ggctgcaaat


 


841
ttgggtaaaa tgaaccgatc ctgccagagc gaaaagctaa ttggctatac gaaaactgtc


 


901
acagaagtcc tggctttcct gcactgctgc ctgaaccctg tgctctacgc ttttattggg


 


961
cagaagttca gaaactactt tctgaagatc ttgaaggacc tgtggtgtgt gagaaggaag


 


1021
tacaagtcct caggcttctc ctgtgccggg aggtactcag aaaacatttc tcggcagacc


 


1081
agtgagaccg cagataacga caatgcgtcg tccttcacta tgtga






The atg start and stop codons are bolded and underlined. The amino acid sequence of human CCR6, provided by Genbank Accession No. AAO92293.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 196).









1
msgesmnfsd vfdssedyfv svntsyysvd semllcslqe vrqfsrlfvp iayslicvfg



 


61
llgnilvvit fafykkarsm tdvyllnmai adilfvltlp fwavshatga wvfsnatckl


 


121
lkgiyainfn cgmllltcis mdryiaivqa tksfrlrsrt lprskiiclv vwglsviiss


 


181
stfvfnqkyn tqgsdvcepk yqtvsepirw kllmlglell fgffiplmfm ifcytfivkt


 


241
lvqaqnskrh kairviiavv lvflacqiph nmvllvtaan lgkmnrscqs ekligytktv


 


301
tevlaflhcc lnpvlyafig qkfrnyflki lkdlwcvrrk ykssgfscag rysenisrqt


 


361
setadndnas sftm






The mRNA sequence encoding human TNFR-1 (tumor necrosis factor receptor 1) provided by Genbank Accession No. NM_001065.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 197).









1
ctcctccagc tcttcctgtc ccgctgttgc aacactgcct cactcttccc ctcccacctt



 


61
ctctcccctc ctctctgctt taattttctc agaattctct ggactgaggc tccagttctg


 


121
gcctttgggg ttcaagatca ctgggaccag gccgtgatct ctatgcccga gtctcaaccc


 


181
tcaactgtca ccccaaggca cttgggacgt cctggacaga ccgagtcccg ggaagcccca


 


241
gcactgccgc tgccacactg ccctgagccc aaatggggga gtgagaggcc atagctgtct


 


301
ggcatgggcc tctccaccgt gcctgacctg ctgctgccac tggtgctcct ggagctgttg


 


361
gtgggaatat acccctcagg ggttattgga ctggtccctc acctagggga cagggagaag


 


421
agagatagtg tgtgtcccca aggaaaatat atccaccctc aaaataattc gatttgctgt


 


481
accaagtgcc acaaaggaac ctacttgtac aatgactgtc caggcccggg gcaggatacg


 


541
gactgcaggg agtgtgagag cggctccttc accgcttcag aaaaccacct cagacactgc


 


601
ctcagctgct ccaaatgccg aaaggaaatg ggtcaggtgg agatctcttc ttgcacagtg


 


661
gaccgggaca ccgtgtgtgg ctgcaggaag aaccagtacc ggcattattg gagtgaaaac


 


721
cttttccagt gcttcaattg cagcctctgc ctcaatggga ccgtgcacct ctcctgccag


 


781
gagaaacaga acaccgtgtg cacctgccat gcaggtttct ttctaagaga aaacgagtgt


 


841
gtctcctgta gtaactgtaa gaaaagcctg gagtgcacga agttgtgcct accccagatt


 


901
gagaatgtta agggcactga ggactcaggc accacagtgc tgttgcccct ggtcattttc


 


961
tttggtcttt gccttttatc cctcctcttc attggtttaa tgtatcgcta ccaacggtgg


 


1021
aagtccaagc tctactccat tgtttgtggg aaatcgacac ctgaaaaaga gggggagctt


 


1081
gaaggaacta ctactaagcc cctggcccca aacccaagct tcagtcccac tccaggcttc


 


1141
acccccaccc tgggcttcag tcccgtgccc agttccacct tcacctccag ctccacctat


 


1201
acccccggtg actgtcccaa ctttgcggct ccccgcagag aggtggcacc accctatcag


 


1261
ggggctgacc ccatccttgc gacagccctc gcctccgacc ccatccccaa cccccttcag


 


1321
aagtgggagg acagcgccca caagccacag agcctagaca ctgatgaccc cgcgacgctg


 


1381
tacgccgtgg tggagaacgt gcccccgttg cgctggaagg aattcgtgcg gcgcctaggg


 


1441
ctgagcgacc acgagatcga tcggctggag ctgcagaacg ggcgctgcct gcgcgaggcg


 


1501
caatacagca tgctggcgac ctggaggcgg cgcacgccgc ggcgcgaggc cacgctggag


 


1561
ctgctgggac gcgtgctccg cgacatggac ctgctgggct gcctggagga catcgaggag


 


1621
gcgctttgcg gccccgccgc cctcccgccc gcgcccagtc ttctcagatg aggctgcgcc


 


1681
cctgcgggca gctctaagga ccgtcctgcg agatcgcctt ccaaccccac ttttttctgg


 


1741
aaaggagggg tcctgcaggg gcaagcagga gctagcagcc gcctacttgg tgctaacccc


 


1801
tcgatgtaca tagcttttct cagctgcctg cgcgccgccg acagtcagcg ctgtgcgcgc


 


1861
ggagagaggt gcgccgtggg ctcaagagcc tgagtgggtg gtttgcgagg atgagggacg


 


1921
ctatgcctca tgcccgtttt gggtgtcctc accagcaagg ctgctcgggg gcccctggtt


 


1981
cgtccctgag cctttttcac agtgcataag cagttttttt tgtttttgtt ttgttttgtt


 


2041
ttgtttttaa atcaatcatg ttacactaat agaaacttgg cactcctgtg ccctctgcct


 


2101
ggacaagcac atagcaagct gaactgtcct aaggcagggg cgagcacgga acaatggggc


 


2161
cttcagctgg agctgtggac ttttgtacat acactaaaat tctgaagtta aagctctgct


 


2221
cttggaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa






The atg start and stop codons are bolded and underlined. The amino acid sequence of human TNFR-1 (tumor necrosis factor receptor 1), provided by Genbank Accession No. NP_001056.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 198).









1
mglstvpdll lplvllellv giypsgvigl vphlgdrekr dsvcpqgkyi hpqnnsicct



 


61
kchkgtylyn dcpgpgqdtd crecesgsft asenhlrhcl scskcrkemg qveissctvd


 


121
rdtvcgcrkn qyrhywsenl fqcfncslcl ngtvhlscqe kqntvctcha gfflrenecv


 


181
scsnckksle ctklclpqie nvkgtedsgt tvllplviff glcllsllfi glmyryqrwk


 


241
sklysivcgk stpekegele gtttkplapn psfsptpgft ptlgfspvps stftssstyt


 


301
pgdcpnfaap rrevappyqg adpilatala sdpipnplqk wedsahkpqs ldtddpatly


 


361
avvenvpplr wkefvrrlgl sdheidrlel qngrclreaq ysmlatwrrr tprreatlel


 


421
lgrvlrdmdl lgcledieea lcgpaalppa psllr


 








Signal peptide AA 1-21; mature peptide AA 22-455).







The mRNA sequence encoding human TNFR-2 provided by Genbank Accession No. M55994.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 199).









1
gaattcggcg cagcggagcc tggagagaag gcgctgggct gcgagggcgc gagggcgcga



 


61
gggcaggggg caaccggacc ccgcccgcac ccatggcgcc cgtcgccgtc tgggccgcgc


 


121
tggccgtcgg actggagctc tgggctgcgg cgcacgcctt gcccgcccag gtggcattta


 


181
caccctacgc cccggagccc gggagcacat gccggctcag agaatactat gaccagacag


 


241
ctcagatgtg ctgcagcaag tgctcgccgg gccaacatgc aaaagtcttc tgtaccaaga


 


301
cctcggacac cgtgtgtgac tcctgtgagg acagcacata cacccagctc tggaactggg


 


361
ttcccgagtg cttgagctgt ggctcccgct gtagctctga ccaggtggaa actcaagcct


 


421
gcactcggga acagaaccgc atctgcacct gcaggcccgg ctggtactgc gcgctgagca


 


481
agcaggaggg gtgccggctg tgcgcgccgc tgcgcaagtg ccgcccgggc ttcggcgtgg


 


541
ccagaccagg aactgaaaca tcagacgtgg tgtgcaagcc ctgtgccccg gggacgttct


 


601
ccaacacgac ttcatccacg gatatttgca ggccccacca gatctgtaac gtggtggcca


 


661
tccctgggaa tgcaagcagg gatgcagtct gcacgtccac gtcccccacc cggagtatgg


 


721
ccccaggggc agtacactta ccccagccag tgtccacacg atcccaacac acgcagccaa


 


781
ctccagaacc cagcactgct ccaagcacct ccttcctgct cccaatgggc cccagccccc


 


841
cagctgaagg gagcactggc gacttcgctc ttccagttgg actgattgtg ggtgtgacag


 


901
ccttgggtct actaataata ggagtggtga actgtgtcat catgacccag gtgaaaaaga


 


961
agcccttgtg cctgcagaga gaagccaagg tgcctcactt gcctgccgat aaggcccggg


 


1021
gtacacaggg ccccgagcag cagcacctgc tgatcacagc gccgagctcc agcagcagct


 


1081
ccctggagag ctcggccagt gcgttggaca gaagggcgcc cactcggaac cagccacagg


 


1141
caccaggcgt ggaggccagt ggggccgggg aggcccgggc cagcaccggg agctcagatt


 


1201
cttcccctgg tggccatggg acccaggtca atgtcacctg catcgtgaac gtctgtagca


 


1261
gctctgacca cagctcacag tgctcctccc aagccagctc cacaatggga gacacagatt


 


1321
ccagcccctc ggagtccccg aaggacgagc aggtcccctt ctccaaggag gaatgtgcct


 


1381
ttcggtcaca gctggagacg ccagagaccc tgctggggag caccgaagag aagcccctgc


 


1441
cccttggagt gcctgatgct gggatgaagc ccagttaacc aggccggtgt gggctgtgtc


 


1501
gtagccaagg tgggctgagc cctggcagga tgaccctgcg aaggggccct ggtccttcca


 


1561
ggcccccacc actaggactc tgaggctctt tctgggccaa gttcctctag tgccctccac


 


1621
agccgcagcc tccctctgac ctgcaggcca agagcagagg cagcgggttg tggaaagcct


 


1681
ctgctgccat ggtgtgtccc tctcggaagg ctggctgggc atggacgttc ggggcatgct


 


1741
ggggcaagtc cctgactctc tgtgacctgc cccgcccagc tgcacctgcc agcctggctt


 


1801
ctggagccct tgggtttttt gtttgtttgt ttgtttgttt gtttgtttct ccccctgggc


 


1861
tctgccccag ctctggcttc cagaaaaccc cagcatcctt ttctgcagag gggctttctg


 


1921
gagaggaggg atgctgcctg agtcacccat gaagacagga cagtgcttca gcctgaggct


 


1981
gagactgcgg gatggtcctg gggctctgtg cagggaggag gtggcagccc tgtagggaac


 


2041
ggggtccttc aagttagctc aggaggcttg gaaagcatca cctcaggcca ggtgcagtcc


 


2101
ctcacgccta tgatcccagc actttgggag gctgaggcgg gtggatcacc tgaggttagg


 


2161
agttcgagac cagcctggcc aacatggtaa aaccccatct ctactaaaaa tacagaaatt


 


2221
agccgggcgt ggtggcgggc acctatagtc ccagctactc agaagcctga ggctgggaaa


 


2281
tcgtttgaac ccgggaagcg gaggttgcag ggagccgaga tcacgccact gcactccagc


 


2341
ctgggcgaca gagcgagagt ctgtctcaaa agaaaaaaaa aaaaaaccga attc






The atg start and stop codons are bolded and underlined. The amino acid sequence of human TNFR-2, provided by Genbank Accession No. AAA36755.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 200).









1
mapvavwaal avglelwaaa halpaqvaft pyapepgstc rlreyydqta qmccskcspg



 


61
qhakvfctkt sdtvcdsced stytqlwnwv peclscgsrc ssdqvetqac treqnrictc


 


121
rpgwycalsk qegcrlcapl rkcrpgfgva rpgtetsdvv ckpcapgtfs nttsstdicr


 


181
phqicnvvai pgnasrdavc tstsptrsma pgavhlpqpv strsqhtqpt pepstapsts


 


241
fllpmgpspp aegstgdfal pvglivgvta lglliigvvn cvimtqvkkk plclqreakv


 


301
phlpadkarg tqgpeqqhll itapssssss lessasaldr raptrnqpqa pgveasgage


 


361
arastgssds spgghgtqvn vtcivnvcss sdhssqcssq asstmgdtds spsespkdeq


 


421
vpfskeecaf rsqletpetl lgsteekplp lgvpdagmkp s 


 








(Signall peptide AA 1-22; mature peptide AA 23-461).







The mRNA sequence encoding human cell death protein (RIP) provided by Genbank Accession No. U25994.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 201).









1
gacgtgaaga gtttaaagaa agagtattca aacgaaaatg cagttgtgaa gagaatgcag



 


61
tctcttcaac ttgattgtgt ggcagtacct tcaagccggt caaattcagc cacagaacag


 


121
cctggttcac tgcacagttc ccagggactt gggatgggtc ctgtggagga gtcctggttt


 


181
gctccttccc tggagcaccc acaagaagag aatgagccca gcctgcagag taaactccaa


 


241
gacgaagcca actaccatct ttatggcagc cgcatggaca ggcagacgaa acagcagccc


 


301
agacagaatg tggcttacaa cagagaggag gaaaggagac gcagggtctc ccatgaccct


 


361
tttgcacagc aaagacctta cgagaatttt cagaatacag agggaaaagg cactgtttat


 


421
tccagtgcag ccagtcatgg taatgcagtg caccagccat cagggctcac cagccaacct


 


481
caagtactgt atcagaacaa tggattatat agctcacatg gctttggaac aagaccactg


 


541
gatccaggaa cagcaggtcc cagagtttgg tacaggccaa ttccaagtca tatgcctagt


 


601
ctgcataata tcccagtgcc tgagaccaac tatctaggaa attctcccac catgccattc


 


661
agctccttgc caccaacaga tgaatctata aaatatacca tatacaatag tactggcatt


 


721
cagattggag cctacaatta tatggagatt ggtgggacga gttcatcact actagacagc


 


781
acaaatacga acttcaaaga agagccagct gctaagtacc aagctatctt tgataatacc


 


841
actagtctga cggataaaca cctggaccca atcagggaaa atctgggaaa gcactggaaa


 


901
aactgtgccc gtaaactggg cttcacacag tctcagattg atgaaattga ccatgactat


 


961
gagcgagatg gactgaaaga aaaggtttac cagatgctcc aaaagtgggt gatgagggaa


 


1021
ggcataaagg gagccacggt ggggaagctg gcccaggcgc tccaccagtg ttccaggatc


 


1081
gaccttctga gcagcttgat ttacgtcagc cagaactaac cctggatggg ctacggcagc


 


1141
tgaagtggac gcctcactta gtggataacc ccagaaagtt ggctgcctca gagcattcag


 


1201
aattctgtcc tcactgatag gggttctgtg tctgcagaaa






The atg start and stop codons are bolded and underlined. The amino acid sequence of human RIP, provided by Genbank Accession No. AAC50137.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 202).









1
dvkslkkeys nenavvkrmq slqldcvavp ssrsnsateq pgslhssqgl gmgpveeswf



 


61
apslehpqee nepslqsklq deanyhlygs rmdrqtkqqp rqnvaynree errrrvshdp


 


121
faqqrpyenf qntegkgtvy ssaashgnav hqpsgltsqp qvlyqnngly sshgfgtrpl


 


181
dpgtagprvw yrpipshmps lhnipvpetn ylgnsptmpf sslpptdesi kytiynstgi


 


241
qigaynymei ggtsssllds tntnfkeepa akyqaifdnt tsltdkhldp irenlgkhwk


 


301
ncarklgftq sqideidhdy erdglkekvy qmlqkwvmre gikgatvgkl aqalhqcsri


 


361
dllssliyvs qn






The mRNA sequence encoding human TRADD provided by Genbank Accession No. NM_003789.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 203).









1
gcacacccgg aagcggcgga gtagagcgga gcctggcggg cgtgggaacc caggccccgc



 


61
cgaggcggcc aggaggtgag atggcagctg ggcaaaatgg gcacgaagag tgggtgggca


 


121
gcgcatacct gtttgtggag tcctcgctgg acaaggtggt cctgtcggat gcctacgcgc


 


181
acccccagca gaaggtggca gtgtacaggg ctctgcaggc tgccttggca gagagcggcg


 


241
ggagcccgga cgtgctgcag atgctgaaga tccaccgcag cgacccgcag ctgatcgtgc


 


301
agctgcgatt ctgcgggcgg cagccctgtg gccgcttcct ccgcgcctac cgcgaggggg


 


361
cgctgcgcgc cgcgctgcag aggagcctgg cggccgcgct cgcccagcac tcggtgccgc


 


421
tgcaactgga gctgcgcgcc ggcgccgagc ggctggacgc tttgctggcg gacgaggagc


 


481
gctgtttgag ttgcatccta gcccagcagc ccgaccggct ccgggatgaa gaactggctg


 


541
agctggagga tgcgctgcga aatctgaagt gcggctcggg ggcccggggt ggcgacgggg


 


601
aggtcgcttc ggcccccttg cagcccccgg tgccctctct gtcggaggtg aagccgccgc


 


661
cgccgccgcc acctgcccag acttttctgt tccagggtca gcctgtagtg aatcggccgc


 


721
tgagcctgaa ggaccaacag acgttcgcgc gctctgtggg tctcaaatgg cgcaaggtgg


 


781
ggcgctcact gcagcgaggc tgccgggcgc tgcgggaccc ggcgctggac tcgctggcct


 


841
acgagtacga gcgcgaggga ctgtacgagc aggccttcca gctgctgcgg cgcttcgtgc


 


901
aggccgaggg ccgccgcgcc acgctgcagc gcctggtgga ggcactcgag gagaacgagc


 


961
tcaccagcct ggcagaggac ttgctgggcc tgaccgatcc caatggcggc ctggcctaga


 


1021
ccaggggtgc agccagcttt tggagaacct ggatggcctt agggttcctt ctgcggctat


 


1081
tgctgaaccc ctgtccatcc acgggaccct gaaactccac ttggcctatc tgctggacct


 


1141
gctggggcag agttgattgc cttccccagg agccagacca ctgggggtgc atcattgggg


 


1201
attctgcctc aggtactttg atagagtgtg gggtgggggg gacctgcttt ggagatcagc


 


1261
ctcaccttct cccatcccag aagcggggct tacagccagc ccttacagtt tcactcatga


 


1321
agcaccttga tctttggtgt cctggacttc atcctgggtg ctgcagatac tgcagtgaag


 


1381
taaaacagga atcaatcttg cctgccccca gctcacactc agcgtgggac cccgaatgtt


 


1441
aagcaatgat aataaagtat aacacggatt ttgatgtgag aaaaaaaaaa aaaaaa






The atg start and stop codons are bolded and underlined. The amino acid sequence of human TRADD, provided by Genbank Accession No. NP_00370.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 204).









1
maagqnghee wvgsaylfve ssldkvvlsd ayahpqqkva vyralqaala esggspdvlq



 


61
mlkihrsdpq livqlrfcgr qpcgrflray regalraalq rslaaalaqh svplqlelra


 


121
gaerldalla deerclscil aqqpdrlrde elaeledalr nlkcgsgarg gdgevasapl


 


181
qppvpslsev kpppppppaq tflfqgqpvv nrplslkdqq tfarsvglkw rkvgrslqrg


 


241
cralrdpald slayeyereg lyeqafqllr rfvqaegrra tlqrlveale eneltslaed


 


301
llgltdpngg la






The mRNA sequence encoding human PADI2 (protein-arginine deiminase type-2) provided by Genbank Accession No. NM_007365.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 205).









1
gcaggctgct ggagaaggcg cacctgctgc aggtgctccc ggccgccccg gaccagcgag



 


61
cgcgggcact gcggcgggga ggatgctgcg cgagcggacc gtgcggctgc agtacgggag


 


121
ccgcgtggag gcggtgtacg tgctgggcac ctacctctgg accgatgtct acagcgcggc


 


181
cccagccggg gcccaaacct tcagcctgaa gcactcggaa cacgtgtggg tggaggtggt


 


241
gcgtgatggg gaggctgagg aggtggccac caatggcaag cagcgctggc ttctctcgcc


 


301
cagcaccacc ctgcgggtca ccatgagcca ggcgagcacc gaggccagca gtgacaaggt


 


361
caccgtcaac tactatgacg aggaagggag cattcccatc gaccaggcgg ggctcttcct


 


421
cacagccatt gagatctccc tggatgtgga cgcagaccgg gatggtgtgg tggagaagaa


 


481
caacccaaag aaggcatcct ggacctgggg ccccgagggc cagggggcca tcctgctggt


 


541
gaactgtgac cgagagacac cctggttgcc caaggaggac tgccgtgatg agaaggtcta


 


601
cagcaaggaa gatctcaagg acatgtccca gatgatcctg cggaccaaag gccccgaccg


 


661
cctccccgcc ggatacgaga tagttctgta catttccatg tcagactcag acaaagtggg


 


721
cgtgttctac gtggagaacc cgttcttcgg ccaacgctat atccacatcc tgggccggcg


 


781
gaagctctac catgtggtca agtacacggg tggctccgcg gagctgctgt tcttcgtgga


 


841
aggcctctgt ttccccgacg agggcttctc aggcctggtc tccatccatg tcagcctgct


 


901
ggagtacatg gcccaggaca ttcccctgac tcccatcttc acggacaccg tgatattccg


 


961
gattgctccg tggatcatga cccccaacat cctgcctccc gtgtcggtgt ttgtgtgctg


 


1021
catgaaggat aattacctgt tcctgaaaga ggtgaagaac cttgtggaga aaaccaactg


 


1081
tgagctgaag gtctgcttcc agtacctaaa ccgaggcgat cgctggatcc aggatgaaat


 


1141
tgagtttggc tacatcgagg ccccccataa aggcttcccc gtggtgctgg actctccccg


 


1201
agatggaaac ctaaaggact tccctgtgaa ggagctcctg ggcccagatt ttggctacgt


 


1261
gacccgggag cccctctttg agtctgtcac cagccttgac tcatttggaa acctggaggt


 


1321
cagtccccca gtgaccgtga acggcaagac atacccgctt ggccgcatcc tcatcgggag


 


1381
cagctttcct ctgtctggtg gtcggaggat gaccaaggtg gtgcgtgact tcctgaaggc


 


1441
ccagcaggtg caggcgcccg tggagctcta ctcagactgg ctgactgtgg gccacgtgga


 


1501
tgagttcatg tcctttgtcc ccatccccgg cacaaagaaa ttcctgctac tcatggccag


 


1561
cacctcggcc tgctacaagc tcttccgaga gaagcagaag gacggccatg gagaggccat


 


1621
catgttcaaa ggcttgggtg ggatgagcag caagcgaatc accatcaaca agattctgtc


 


1681
caacgagagc cttgtgcagg agaacctgta cttccagcgc tgcctagact ggaaccgtga


 


1741
catcctcaag aaggagctgg gactgacaga gcaggacatc attgacctgc ccgctctgtt


 


1801
caagatggac gaggaccacc gtgccagagc cttcttccca aacatggtga acatgatcgt


 


1861
gctggacaag gacctgggca tccccaagcc attcgggcca caggttgagg aggaatgctg


 


1921
cctggagatg cacgtgcgtg gcctcctgga gcccctgggc ctcgaatgca ccttcatcga


 


1981
cgacatttct gcctaccaca aatttctggg ggaagtccac tgtggcacca acgtccgcag


 


2041
gaagcccttc accttcaagt ggtggcacat ggtgccctga cctgccaggg gccctggcgt


 


2101
ttgcctcctt cgcttagttc tccagaccct ccctcacacg cccagagcct tctgctgaca


 


2161
tggactggac agccccgctg ggagaccttt gggacgtggg gtggaatttg gggtatctgt


 


2221
gccttgccct ccctgagagg ggcctcagtg tcctctgaag ccatccccag tgagcctcga


 


2281
ctctgtccct gctgaaaata gctgggccag tgtctctgta gccctgacat aaggaacaga


 


2341
acacaacaaa acacagcaaa ccatgtgccc aaactgctcc ccaaagaatt ttgagtctct


 


2401
aatctgacac tgaatgaggg gagaagggaa ggagattctg ggattgccag ttcttccagc


 


2461
agccatgctc tgaaaatcaa ggtagaatcc atggaaaggg accccaggac cccgggaccc


 


2521
tagacgtatc ttgaactgcc atcgtcattt caaatacatc tccctcaggg tttccaggtg


 


2581
gccaccccca attattcatt ccttaccaac ctctcaaatc ctcttggctt tctctctgca


 


2641
gtgtggacac tgttggctag tcctccccac tccctgaggg tccagtaagt tagcttagaa


 


2701
ccttcctgga aacatttcat ctgagcaggt ttccccacgt gtgggatgct ccttttgcct


 


2761
catctgtctc agggatgcag gctcccccgc atgcatgggg atttctcccc agaccagcat


 


2821
acttgtgacc tgagagttca atgcgtaaag atgcccctgg tcagccatat ccatcttctc


 


2881
ttgcctggtc cttgattctc tggccgctcc ctgaccttcc tccttccact gccttgactt


 


2941
tcttcctttt tattcctggt gccatctgtc caggcagcta gacaagaact tgttcgccag


 


3001
cagccagatt caggccttcc caggggcata ataagtgacc agcccctcct ctccggacat


 


3061
cagatccaac acataaggac cctggcctac cctccagccc aacagccagt tctgggtcag


 


3121
ctgccaactt aggggtggtt tgattatccc attgaaattc accagtgcct ttgccaaaga


 


3181
ccctctcatt tggacatacc cagattcatt ccctggctcc aactgaaaag actcagtttc


 


3241
aatcgttaaa agttccttta gggccagaag aataaatgaa ttataatccc attttgaaga


 


3301
accgatttat aaccaatgaa aaggttataa tgtaatttat attcttggag gaacaagatt


 


3361
ttcatttggg attatttcct tcaaccattc aacaaacatt tgttgtatgc cactaagcgc


 


3421
caggcacggc gttgggctct gcaaacacag tggttagtag cagtctggac ctggtcccta


 


3481
ctggcatgga acccatcact ccccaacatg caaagcccac atttaaaggc cagcctctgc


 


3541
cccttcagtg atgcgctctt tagaaatgcc agtccactat attcagaaat ccgcagggca


 


3601
caaaacttcc agcaagtcac tgttgtggtg aaatgggcag tgggggtggg gggtcttctt


 


3661
taaacaggcc cccttcccat ctacctagcc agtacccatc caatgagtcc ccagagcctc


 


3721
cagaagctgt tgtctcctct ctggggacag cagctcctgc ctttggaggc caaagcccca


 


3781
gatctctcca gccccagagc tgaaaacacc aagtgcctat ttgagggtgt ctgtctggag


 


3841
acttagagtt tgtcatgtgt gtgtgtgtgt ttggttaatg tgggtttatg ggttttcttt


 


3901
cttttttttc tttttttttt tagtctacat tagggggaag tgagcgcctc ccatgtgcag


 


3961
acagtgtgtc tttatagatt tttctaaggc tttccccaat gatgtcggta atttctgatg


 


4021
tttctgaagt tcccaggact cacacacccg ttcccatctc acttgcccac ccagtgtgac


 


4081
aaccctcggt gtggatatac ccccgtggac tcatggctct tccccacccc cactttctat


 


4141
aaatgtaggc ctagaatacg cttctctgtt gcaaaactca gctaagttcc tgcttccacc


 


4201
ttgatgttga aatatcttat gtaagagggc aggggatgtc gtgaagatgg caagaagaac


 


4261
acagtttcaa atttctggaa aagagcctgt ggtggagatc taaagatgtt tagggaagag


 


4321
ctcgactaaa gaacaatgaa ataaatggtc caaggggaag tca






The atg start and stop codons are bolded and underlined. The amino acid sequence of human PADI2 (protein-arginine deiminase type-2), provided by Genbank Accession No. NP_031391.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 206).









1
mlrertvrlq ygsrveavyv lgtylwtdvy saapagaqtf slkhsehvwv evvrdgeaee



 


61
vatngkqrwl lspsttlrvt msqasteass dkvtvnyyde egsipidqag lfltaieisl


 


121
dvdadrdgvv eknnpkkasw twgpegqgai llvncdretp wlpkedcrde kvyskedlkd


 


181
msqmilrtkg pdrlpagyei vlyismsdsd kvgvfyvenp ffgqryihil grrklyhvvk


 


241
ytggsaellf fveglcfpde gfsglvsihv slleymaqdi pltpiftdtv ifriapwimt


 


301
pnilppvsvf vccmkdnylf lkevknlvek tncelkvcfq ylnrgdrwiq deiefgyiea


 


361
phkgfpvvld sprdgnlkdf pvkellgpdf gyvtreplfe svtsldsfgn levsppvtvn


 


421
gktyplgril igssfplsgg rrmtkvvrdf lkaqqvqapv elysdwltvg hvdefmsfvp


 


481
ipgtkkflll mastsacykl frekqkdghg eaimfkglgg msskritink ilsneslvqe


 


541
nlyfqrcldw nrdilkkelg lteqdiidlp alfkmdedhr araffpnmvn mivldkdlgi


 


601
pkpfgpqvee ecclemhvrg lleplglect fiddisayhk flgevhcgtn vrrkpftfkw


 


661
whmvp






The mRNA sequence encoding human PAD3 (PADI3) provided by Genbank Accession No. NM_016233.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 207).









1
agtgttgggg ttggcggcca cagctaagtc caacaccagc atgtcgctgc agagaatcgt



 


61
gcgtgtgtcc ctggagcatc ccaccagcgc ggtgtgtgtg gctggcgtgg agaccctcgt


 


121
ggacatttat gggtcagtgc ctgagggcac agaaatgttt gaggtctatg ggacgcctgg


 


181
cgtggacatc tacatctctc ccaacatgga gaggggccgg gagcgtgcag acaccaggcg


 


241
gtggcgcttt gacgcgactt tggagatcat cgtggtcatg aactccccca gcaatgacct


 


301
caacgacagc catgttcaga tttcctacca ctccagccat gagcctctgc ccctggccta


 


361
tgcggtgctc tacctcacct gtgttgacat ctctctggat tgcgacctga actgtgaggg


 


421
aaggcaggac aggaactttg tagacaagcg gcagtgggtc tgggggccca gtgggtatgg


 


481
cggcatcttg ctggtgaact gtgaccgtga tgatccgagc tgtgatgtcc aggacaattg


 


541
tgaccagcac gtgcactgcc tgcaagacct ggaagacatg tctgtcatgg tcctgcggac


 


601
gcagggccct gcagccctct ttgatgacca caaacttgtc ctccatacct ccagctatga


 


661
tgccaaacgg gcacaggtct tccacatctg cggtcctgag gatgtgtgtg aggcctatag


 


721
gcatgtgctg ggccaagata aggtgtccta tgaggtaccc cgcttgcatg gggatgagga


 


781
gcgcttcttc gtggaaggcc tgtccttccc tgatgccggc ttcacaggac tcatctcctt


 


841
ccatgtcact ctgctggacg actccaacga ggatttctcg gcatccccta tcttcactga


 


901
cactgtggtg ttccgagtgg caccctggat catgacgccc agcactctgc cacccctaga


 


961
ggtgtatgtg tgccgtgtga ggaacaacac gtgttttgtg gatgcggtgg cagagctggc


 


1021
caggaaggcc ggctgcaagc tgaccatctg cccacaggcc gagaaccgca acgaccgctg


 


1081
gatccaggat gagatggagc tgggctacgt tcaggcgccg cacaagaccc tcccggtggt


 


1141
ctttgactcc ccaaggaatg gggaactgca ggatttccct tacaaaagaa tcctgggtcc


 


1201
agattttggt tacgtgactc gggaaccacg cgacaggtct gtgagtggcc tggactcctt


 


1261
tgggaacctg gaggtcagcc ctccagtggt ggccaatggg aaagagtacc ccctggggag


 


1321
gatcctcatt gggggcaacc tgcctgggtc aagtggccgc agggtcaccc aggtggtgcg


 


1381
ggacttcctc catgcccaga aggtgcagcc ccccgtggag ctctttgtgg actggttggc


 


1441
cgtgggccat gtggatgagt ttctgagctt tgtccctgcc cccgatggga agggcttccg


 


1501
gatgctcctg gccagccctg gggcctgctt caagctcttc caggaaaagc agaagtgtgg


 


1561
ccacgggagg gccctcctgt tccagggggt tgttgatgat gagcaggtca agaccatctc


 


1621
catcaaccag gtgctctcca ataaagacct catcaactac aataagtttg tgcagagctg


 


1681
catcgactgg aaccgtgagg tgctgaagcg ggagctgggc ctggcagagt gtgacatcat


 


1741
tgacatccca cagctcttca agaccgagag gaaaaaagca acggccttct tccctgactt


 


1801
ggtgaacatg ctggtgctgg ggaagcacct gggcatcccc aagccctttg ggcccatcat


 


1861
caatggctgc tgctgcctgg aggagaaggt gcggtccctg ctggagccgc tgggcctcca


 


1921
ctgcaccttc attgatgact tcactccata ccacatgctg catggggagg tgcactgtgg


 


1981
caccaatgtg tgcagaaagc ccttctcttt caagtggtgg aacatggtgc cctgagacag


 


2041
ctcccaccca ccatcctgtc cccctggggc gggcattggc ccaggtggtg gagacagaga


 


2101
caggcccctg aacgataagc accaagagac cccaaggctc cagatggaac actgagggtg


 


2161
accgtccctc tcagaagcct tttccctgga agtgtccatg cctcacctgc aacccatgtg


 


2221
gttctcagac ttgaatcttc tcggcccccc aaaaagaagg acctcatttc ttatagcctc


 


2281
tcctgtgatt caacacaacc catggagatg tccccttctc actctgaaat catccatttg


 


2341
gggacaaatc cacattgggg tctagaaaca tccacgtatc tcatcagcca tcttgtcctg


 


2401
tgcatcctaa cagaggaagg atccatgatt ctgctttggt ccaattgctt cctctctgca


 


2461
gaggaacaac cctaaaacca gaccactcca cgcaggacag gcaggagaga ttcttcctaa


 


2521
agcctccccc ataaaaaggg agctgtggat ccacttagat cagggcggaa ccatctttca


 


2581
cccggccaag ctcctgccca gatgttgacc ctcacccagc gtgagctgtc acatagtagg


 


2641
agcttctaga tgcatgtgga agcaatgaga gttgtccctt agccttataa actccccatg


 


2701
atctgacatg cagaaatcca gccttgtcca gaatcctcct ggaatttctt ggagacgaaa


 


2761
gtatctgggg gattgttggg tactagggag actgggtaca agggtgaaaa gtagttccca


 


2821
taatacacat ggttgactat ggtgatccac cttgtgatgg ttaatattag gtgtctggag


 


2881
aaggttgctt cattggccct gggacttctc tctgcaggag gagagaacgc tgcctctcct


 


2941
ctggattggt ctcaggctct ctgttggcct ttggtcagcg tttccacatc ctgctctgct


 


3001
gcaggagagg gggctaaggg gctggatcca ccaaggcagc tcacagcggg aaaactctgg


 


3061
gaatgaacca ctgaattcag gggatggggg tgggggggcg gttctcgagg tgtgtgccag


 


3121
ctacacgtgt gttctgtatg ggtccagctg cgtttccatc actcgctaat aaatcaacag


 


3181
aaacacaaa






The atg start and stop codons are bolded and underlined. The amino acid sequence of human PADI3 (PAD3), provided by Genbank Accession No. NP_057317.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 208).









1
mslqrivrvs lehptsavcv agvetlvdiy gsvpegtemf evygtpgvdi yispnmergr



 


61
eradtrrwrf datleiivvm nspsndlnds hvqisyhssh eplplayavl yltcvdisld


 


121
cdlncegrqd rnfvdkrqwv wgpsgyggil lvncdrddps cdvqdncdqh vhclqdledm


 


181
svmvlrtqgp aalfddhklv lhtssydakr aqvfhicgpe dvceayrhvl gqdkvsyevp


 


241
rlhgdeerff veglsfpdag ftglisfhvt llddsnedfs aspiftdtvv frvapwimtp


 


301
stlpplevyv crvrnntcfv davaelarka gcklticpqa enrndrwiqd emelgyvqap


 


361
hktlpvvfds prngelqdfp ykrilgpdfg yvtreprdrs vsgldsfgnl evsppvvang


 


421
keyplgrili ggnlpgssgr rvtqvvrdfl haqkvqppve lfvdwlavgh vdeflsfvpa


 


481
pdgkgfrmll aspgacfklf qekqkcghgr allfqgvvdd eqvktisinq vlsnkdliny


 


541
nkfvqscidw nrevlkrelg laecdiidip qlfkterkka taffpdlvnm lvlgkhlgip


 


601
kpfgpiingc ccleekvrsl leplglhctf iddftpyhml hgevhcgtnv crkpfsfkww


 


661
nmvp






The mRNA sequence encoding human FOXP3 provided by Genbank Accession No. EF534714.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 209).









1
atgcccaacc ccaggcctgg caagccctcg gccccttcct tggcccttgg cccatcccca



 


61
ggagcctcgc ccagctggag ggctgcaccc aaagcctcag acctgctggg ggcccggggc


 


121
ccagggggaa ccttccaggg ccgagatctt cgaggcgggg cccatgcctc ctcttcttcc


 


181
ttgaacccca tgccaccatc gcagctgcag ctgcccacac tgcccctagt catggtggca


 


241
ccctccgggg cacggctggg ccccttgccc cacttacagg cactcctcca ggacaggcca


 


301
catttcatgc accagctctc aacggtggat gcccacgccc ggacccctgt gctgcaggtg


 


361
caccccctgg agagcccagc catgatcagc ctcacaccac ccaccaccgc cactggggtc


 


421
ttctccctca aggcccggcc tggcctccca cctgggatca acgtggccag cctggaatgg


 


481
gtgtccaggg agccggcact gctctgcacc ttcccaaatc ccagtgcacc caggaaggac


 


541
agcacccttt cggctgtgcc ccagagctcc tacccactgc tggcaaatgg tgtctgcaag


 


601
tggcccggat gtgagaaggt cttcgaagag ccagaggact tcctcaagca ctgccaggcg


 


661
gaccatcttc tggatgagaa gggcagggca caatgtctcc tccagagaga gatggtacag


 


721
tctctggagc agcagctggt gctggagaag gagaagctga gtgccatgca ggcccacctg


 


781
gctgggaaaa tggcactgac caaggcttca tctgtggcat catccgacaa gggctcctgc


 


841
tgcatcgtag ctgctggcag ccaaggccct gtcgtcccag cctggtctgg cccccgggag


 


901
gcccctgaca gcctgtttgc tgtccggagg cacctgtggg gtagccatgg aaacagcaca


 


961
ttcccagagt tcctccacaa catggactac ttcaagttcc acaacatgcg accccctttc


 


1021
acctacgcca cgctcatccg ctgggccatc ctggaggctc cagagaagca gcggacactc


 


1081
aatgagatct accactggtt cacacgcatg tttgccttct tcagaaacca tcctgccacc


 


1141
tggaagaacg ccatccgcca caacctgagt ctgcacaagt gctttgtgcg ggtggagagc


 


1201
gagaaggggg ctgtgtggac cgtggatgag ctggagttcc gcaagaaacg gagccagagg


 


1261
cccagcaggt gttccaaccc tacacctggc ccctga






The atg start and stop codons are bolded and underlined. The amino acid sequence of human FOXP3, provided by Genbank Accession No. ABQ15210.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 210).









1
mpnprpgkps apslalgpsp gaspswraap kasdllgarg pggtfqgrdl rggahassss



 


61
lnpmppsqlq lptlplvmva psgarlgplp hlqallqdrp hfmhqlstvd ahartpvlqv


 


121
hplespamis ltppttatgv fslkarpglp pginvaslew vsrepallct fpnpsaprkd


 


181
stlsavpqss ypllangvck wpgcekvfee pedflkhcqa dhlldekgra qcllqremvq


 


241
sleqqlvlek eklsamqahl agkmaltkas svassdkgsc civaagsqgp vvpawsgpre


 


301
apdslfavrr hlwgshgnst fpeflhnmdy fkfhnmrppf tyatlirwai leapekqrtl


 


361
neiyhwftrm faffrnhpat wknairhnls lhkcfvrves ekgavwtvde lefrkkrsqr


 


421
psrcsnptpg p






The mRNA sequence encoding human IL2RA (CD-25) provided by Genbank Accession No. NM_000417.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 211).








(SEQ ID NO: 211)










1
ggcagtttcc tggctgaaca cgccagccca atacttaaag agagcaactc ctgactccga



 


61
tagagactgg atggacccac aagggtgaca gcccaggcgg accgatcttc ccatcccaca


 


121
tcctccggcg cgatgccaaa aagaggctga cggcaactgg gccttctgca gagaaagacc


 


181
tccgcttcac tgccccggct ggtcccaagg gtcaggaaga tggattcata cctgctgatg


 


241
tggggactgc tcacgttcat catggtgcct ggctgccagg cagagctctg tgacgatgac


 


301
ccgccagaga tcccacacgc cacattcaaa gccatggcct acaaggaagg aaccatgttg


 


361
aactgtgaat gcaagagagg tttccgcaga ataaaaagcg ggtcactcta tatgctctgt


 


421
acaggaaact ctagccactc gtcctgggac aaccaatgtc aatgcacaag ctctgccact


 


481
cggaacacaa cgaaacaagt gacacctcaa cctgaagaac agaaagaaag gaaaaccaca


 


541
gaaatgcaaa gtccaatgca gccagtggac caagcgagcc ttccaggtca ctgcagggaa


 


601
cctccaccat gggaaaatga agccacagag agaatttatc atttcgtggt ggggcagatg


 


661
gtttattatc agtgcgtcca gggatacagg gctctacaca gaggtcctgc tgagagcgtc


 


721
tgcaaaatga cccacgggaa gacaaggtgg acccagcccc agctcatatg cacaggtgaa


 


781
atggagacca gtcagtttcc aggtgaagag aagcctcagg caagccccga aggccgtcct


 


841
gagagtgaga cttcctgcct cgtcacaaca acagattttc aaatacagac agaaatggct


 


901
gcaaccatgg agacgtccat atttacaaca gagtaccagg tagcagtggc cggctgtgtt


 


961
ttcctgctga tcagcgtcct cctcctgagt gggctcacct ggcagcggag acagaggaag


 


1021
agtagaagaa caatctagaa aaccaaaaga acaagaattt cttggtaaga agccgggaac


 


1081
agacaacaga agtcatgaag cccaagtgaa atcaaaggtg ctaaatggtc gcccaggaga


 


1141
catccgttgt gcttgcctgc gttttggaag ctctgaagtc acatcacagg acacggggca


 


1201
gtggcaacct tgtctctatg ccagctcagt cccatcagag agcgagcgct acccacttct


 


1261
aaatagcaat ttcgccgttg aagaggaagg gcaaaaccac tagaactctc catcttattt


 


1321
tcatgtatat gtgttcatta aagcatgaat ggtatggaac tctctccacc ctatatgtag


 


1381
tataaagaaa agtaggttta cattcatctc attccaactt cccagttcag gagtcccaag


 


1441
gaaagcccca gcactaacgt aaatacacaa cacacacact ctaccctata caactggaca


 


1501
ttgtctgcgt ggttcctttc tcagccgctt ctgactgctg attctcccgt tcacgttgcc


 


1561
taataaacat ccttcaagaa ctctgggctg ctacccagaa atcattttac ccttggctca


 


1621
atcctctaag ctaaccccct tctactgagc cttcagtctt gaatttctaa aaaacagagg


 


1681
ccatggcaga ataatctttg ggtaacttca aaacggggca gccaaaccca tgaggcaatg


 


1741
tcaggaacag aaggatgaat gaggtcccag gcagagaatc atacttagca aagttttacc


 


1801
tgtgcgttac taattggcct ctttaagagt tagtttcttt gggattgcta tgaatgatac


 


1861
cctgaatttg gcctgcacta atttgatgtt tacaggtgga cacacaaggt gcaaatcaat


 


1921
gcgtacgttt cctgagaagt gtctaaaaac accaaaaagg gatccgtaca ttcaatgttt


 


1981
atgcaaggaa ggaaagaaag aaggaagtga agagggagaa gggatggagg tcacactggt


 


2041
agaacgtaac cacggaaaag agcgcatcag gcctggcacg gtggctcagg cctataaccc


 


2101
cagctcccta ggagaccaag gcgggagcat ctcttgaggc caggagtttg agaccagcct


 


2161
gggcagcata gcaagacaca tccctacaaa aaattagaaa ttggctggat gtggtggcat


 


2221
acgcctgtag tcctagccac tcaggaggct gaggcaggag gattgcttga gcccaggagt


 


2281
tcgaggctgc agtcagtcat gatggcacca ctgcactcca gcctgggcaa cagagcaaga


 


2341
tcctgtcttt aaggaaaaaa agacaagatg agcataccag cagtccttga acattatcaa


 


2401
aaagttcagc atattagaat caccgggagg ccttgttaaa agagttcgct gggcccatct


 


2461
tcagagtctc tgagttgttg gtctggaata gagccaaatg ttttgtgtgt ctaacaattc


 


2521
ccaggtgctg ttgctgctgc tactattcca ggaacacact ttgagaacca ttgtgttatt


 


2581
gctctgcacg cccacccact ctcaactccc acgaaaaaaa tcaacttcca gagctaagat


 


2641
ttcggtggaa gtcctggttc catatctggt gcaagatctc ccctcacgaa tcagttgagt


 


2701
caacattcta gctcaacaac atcacacgat taacattaac gaaaattatt catttgggaa


 


2761
actatcagcc agttttcact tctgaagggg caggagagtg ttatgagaaa tcacggcagt


 


2821
tttcagcagg gtccagattc agattaaata actattttct gtcatttctg tgaccaacca


 


2881
catacaaaca gactcatctg tgcactctcc ccctccccct tcaggtatat gttttctgag


 


2941
taaagttgaa aagaatctca gaccagaaaa tatagatata tatttaaatc ttacttgagt


 


3001
agaactgatt acgacttttg ggtgttgagg ggtctataag atcaaaactt ttccatgata


 


3061
atactaagat gttatcgacc atttatctgt ccttctctca aaagtgtatg gtggaatttt


 


3121
ccagaagcta tgtgatacgt gatgatgtca tcactctgct gttaacatat aataaattta


 


3181
ttgctattgt ttataaaaga ataaatgata tttttt






The atg start and stop codons are bolded and underlined. The amino acid sequence of human IL2RA (CD-25), provided by Genbank Accession No. NP_000408, is incorporated herein by reference, and is shown below (SEQ ID NO: 212).








(SEQ ID NO: 212)










1
mdsyllmwgl ltfimvpgcq aelcdddppe iphatfkama ykegtmlnce ckrgfrriks



 


61
gslymlctgn sshsswdnqc qctssatrnt tkqvtpqpee qkerkttemq spmqpvdqas


 


121
lpghcreppp weneateriy hfvvgqmvyy qcvqgyralh rgpaesvckm thgktrwtqp


 


181
qlictgemet sqfpgeekpq aspegrpese tsclvtttdf qiqtemaatm etsiftteyq


 


241
vavagcvfll isvlllsglt wqrrqrksrr ti


 








(Signal protein AA 1-21; mature protein AA 22-272).







The mRNA sequence encoding human FAP (fibroblast activation protein) provided by Genbank Accession No. NM_001291807.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 213).









1
aagaacgccc ccaaaatctg tttctaattt tacagaaatc ttttgaaact tggcacggta



 


61
ttcaaaagtc cgtggaaaga aaaaaacctt gtcctggctt cagcttccaa ctacaaagac


 


121
agacttggtc cttttcaacg gttttcacag atccagtgac ccacgctctg aagacagaat


 


181
tagctaactt tcaaaaacat ctggaaaaat gaagacttgg gtaaaaatcg tatttggagt


 


241
tgccacctct gctgtgcttg ccttattggt gatgtgcatt gtcttacgcc cttcaagagt


 


301
tcataactct gaagaaaata caatgagagc actcacactg aaggatattt taaatggaac


 


361
attttcttat aaaacatttt ttccaaactg gatttcagga caagaatatc ttcatcaatc


 


421
tgcagataac aatatagtac tttataatat tgaaacagga caatcatata ccattttgag


 


481
taatagaacc atgctttgga gatactctta cacagcaaca tattacatct atgaccttag


 


541
caatggagaa tttgtaagag gaaatgagct tcctcgtcca attcagtatt tatgctggtc


 


601
gcctgttggg agtaaattag catatgtcta tcaaaacaat atctatttga aacaaagacc


 


661
aggagatcca ccttttcaaa taacatttaa tggaagagaa aataaaatat ttaatggaat


 


721
cccagactgg gtttatgaag aggaaatgct tgctacaaaa tatgctctct ggtggtctcc


 


781
taatggaaaa tttttggcat atgcggaatt taatgatacg gatataccag ttattgccta


 


841
ttcctattat ggcgatgaac aatatcctag aacaataaat attccatacc caaaggctgg


 


901
agctaagaat cccgttgttc ggatatttat tatcgatacc acttaccctg cgtatgtagg


 


961
tccccaggaa gtgcctgttc cagcaatgat agcctcaagt gattattatt tcagttggct


 


1021
cacgtgggtt actgatgaac gagtatgttt gcagtggcta aaaagagtcc agaatgtttc


 


1081
ggtcctgtct atatgtgact tcagggaaga ctggcagaca tgggattgtc caaagaccca


 


1141
ggagcatata gaagaaagca gaactggatg ggctggtgga ttctttgttt caacaccagt


 


1201
tttcagctat gatgccattt cgtactacaa aatatttagt gacaaggatg gctacaaaca


 


1261
tattcactat atcaaagaca ctgtggaaaa tgctattcaa attacaagtg gcaagtggga


 


1321
ggccataaat atattcagag taacacagga ttcactgttt tattctagca atgaatttga


 


1381
agaataccct ggaagaagaa acatctacag aattagcatt ggaagctatc ctccaagcaa


 


1441
gaagtgtgtt acttgccatc taaggaaaga aaggtgccaa tattacacag caagtttcag


 


1501
cgactacgcc aagtactatg cacttgtctg ctacggccca ggcatcccca tttccaccct


 


1561
tcatgatgga cgcactgatc aagaaattaa aatcctggaa gaaaacaagg aattggaaaa


 


1621
tgctttgaaa aatatccagc tgcctaaaga ggaaattaag aaacttgaag tagatgaaat


 


1681
tactttatgg tacaagatga ttcttcctcc tcaatttgac agatcaaaga agtatccctt


 


1741
gctaattcaa gtgtatggtg gtccctgcag tcagagtgta aggtctgtat ttgctgttaa


 


1801
ttggatatct tatcttgcaa gtaaggaagg gatggtcatt gccttggtgg atggtcgagg


 


1861
aacagctttc caaggtgaca aactcctcta tgcagtgtat cgaaagctgg gtgtttatga


 


1921
agttgaagac cagattacag ctgtcagaaa attcatagaa atgggtttca ttgatgaaaa


 


1981
aagaatagcc atatggggct ggtcctatgg aggatacgtt tcatcactgg cccttgcatc


 


2041
tggaactggt cttttcaaat gtggtatagc agtggctcca gtctccagct gggaatatta


 


2101
cgcgtctgtc tacacagaga gattcatggg tctcccaaca aaggatgata atcttgagca


 


2161
ctataagaat tcaactgtga tggcaagagc agaatatttc agaaatgtag actatcttct


 


2221
catccacgga acagcagatg ataatgtgca ctttcaaaac tcagcacaga ttgctaaagc


 


2281
tctggttaat gcacaagtgg atttccaggc aatgtggtac tctgaccaga accacggctt


 


2341
atccggcctg tccacgaacc acttatacac ccacatgacc cacttcctaa agcagtgttt


 


2401
ctctttgtca gactaaaaac gatgcagatg caagcctgta tcagaatctg aaaaccttat


 


2461
ataaacccct cagacagttt gcttatttta ttttttatgt tgtaaaatgc tagtataaac


 


2521
aaacaaatta atgttgttct aaaggctgtt aaaaaaaaga tgaggactca gaagttcaag


 


2581
ctaaatattg tttacatttt ctggtactct gtgaaagaag agaaaaggga gtcatgcatt


 


2641
ttgctttgga cacagtgttt tatcacctgt tcatttgaag aaaaataata aagtcagaag


 


2701
ttcaagtgct aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa






The atg start and stop codons are bolded and underlined. The amino acid sequence of human FAP (fibroblast activation protein), provided by Genbank Accession No. NP_001278736.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 214).









1
mktwvkivfg vatsavlall vmcivlrpsr vhnseentmr altlkdilng tfsyktffpn



 


61
wisgqeylhq sadnnivlyn ietgqsytil snrtmlwrys ytatyyiydl sngefvrgne


 


121
lprpiqylcw spvgsklayv yqnniylkqr pgdppfqitf ngrenkifng ipdwvyeeem


 


181
latkyalwws pngkflayae fndtdipvia ysyygdeqyp rtinipypka gaknpvvrif


 


241
iidttypayv gpqevpvpam iassdyyfsw ltwvtdervc lqwlkrvqnv svlsicdfre


 


301
dwqtwdcpkt qehieesrtg waggffvstp vfsydaisyy kifsdkdgyk hihyikdtve


 


361
naiqitsgkw eainifrvtq dslfyssnef eeypgrrniy risigsypps kkcvtchlrk


 


421
ercqyytasf sdyakyyalv cygpgipist lhdgrtdqei kileenkele nalkniqlpk


 


481
eeikklevde itlwykmilp pqfdrskkyp lliqvyggpc sqsvrsvfav nwisylaske


 


541
gmvialvdgr gtafqgdkll yavyrklgvy evedqitavr kfiemgfide kriaiwgwsy


 


601
ggyvsslala sgtglfkcgi avapvsswey yasvyterfm glptkddnle hyknstvmar


 


661
aeyfrnvdyl lihgtaddnv hfqnsaqiak alvnaqvdfq amwysdqnhg lsglstnhly


 


721
thmthflkqc fslsd






The mRNA sequence encoding human DPP4 (dipeptidyl peptidase 4) provided by Genbank Accession No. NM_001935.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 215).









1
ctttcactgg caagagacgg agtcctgggt ttcagttcca gttgcctgcg gtgggctgtg



 


61
tgagtttgcc aaagtcccct gccctctctg ggtctcggtt ccctcgcctg tccacgtgag


 


121
gttggaggag ctgaacgccg acgtcatttt tagctaagag ggagcagggt ccccgagtcg


 


181
ccggcccagg gtctgcgcat ccgaggccgc gcgccctttc ccctccccca cggctcctcc


 


241
gggccccgca ctctgcgccc cggctgccgc ccagcgccct acaccgccct cagggggccc


 


301
tcgcgggctc cccccggccg ggatgccagt gccccgcgcc acgcgcgcct gctcccgcgc


 


361
cgcctgccct gcagcctgcc cgcggcgcct ttatacccag cgggctcggc gctcactaat


 


421
gtttaactcg gggccgaaac ttgccagcgg cgagtgactc caccgcccgg agcagcggtg


 


481
caggacgcgc gtctccgccg cccgcggtga cttctgcctg cgctccttct ctgaacgctc


 


541
acttccgagg agacgccgac gatgaagaca ccgtggaagg ttcttctggg actgctgggt


 


601
gctgctgcgc ttgtcaccat catcaccgtg cccgtggttc tgctgaacaa aggcacagat


 


661
gatgctacag ctgacagtcg caaaacttac actctaactg attacttaaa aaatacttat


 


721
agactgaagt tatactcctt aagatggatt tcagatcatg aatatctcta caaacaagaa


 


781
aataatatct tggtattcaa tgctgaatat ggaaacagct cagttttctt ggagaacagt


 


841
acatttgatg agtttggaca ttctatcaat gattattcaa tatctcctga tgggcagttt


 


901
attctcttag aatacaacta cgtgaagcaa tggaggcatt cctacacagc ttcatatgac


 


961
atttatgatt taaataaaag gcagctgatt acagaagaga ggattccaaa caacacacag


 


1021
tgggtcacat ggtcaccagt gggtcataaa ttggcatatg tttggaacaa tgacatttat


 


1081
gttaaaattg aaccaaattt accaagttac agaatcacat ggacggggaa agaagatata


 


1141
atatataatg gaataactga ctgggtttat gaagaggaag tcttcagtgc ctactctgct


 


1201
ctgtggtggt ctccaaacgg cactttttta gcatatgccc aatttaacga cacagaagtc


 


1261
ccacttattg aatactcctt ctactctgat gagtcactgc agtacccaaa gactgtacgg


 


1321
gttccatatc caaaggcagg agctgtgaat ccaactgtaa agttctttgt tgtaaataca


 


1381
gactctctca gctcagtcac caatgcaact tccatacaaa tcactgctcc tgcttctatg


 


1441
ttgatagggg atcactactt gtgtgatgtg acatgggcaa cacaagaaag aatttctttg


 


1501
cagtggctca ggaggattca gaactattcg gtcatggata tttgtgacta tgatgaatcc


 


1561
agtggaagat ggaactgctt agtggcacgg caacacattg aaatgagtac tactggctgg


 


1621
gttggaagat ttaggccttc agaacctcat tttacccttg atggtaatag cttctacaag


 


1681
atcatcagca atgaagaagg ttacagacac atttgctatt tccaaataga taaaaaagac


 


1741
tgcacattta ttacaaaagg cacctgggaa gtcatcggga tagaagctct aaccagtgat


 


1801
tatctatact acattagtaa tgaatataaa ggaatgccag gaggaaggaa tctttataaa


 


1861
atccaactta gtgactatac aaaagtgaca tgcctcagtt gtgagctgaa tccggaaagg


 


1921
tgtcagtact attctgtgtc attcagtaaa gaggcgaagt attatcagct gagatgttcc


 


1981
ggtcctggtc tgcccctcta tactctacac agcagcgtga atgataaagg gctgagagtc


 


2041
ctggaagaca attcagcttt ggataaaatg ctgcagaatg tccagatgcc ctccaaaaaa


 


2101
ctggacttca ttattttgaa tgaaacaaaa ttttggtatc agatgatctt gcctcctcat


 


2161
tttgataaat ccaagaaata tcctctacta ttagatgtgt atgcaggccc atgtagtcaa


 


2221
aaagcagaca ctgtcttcag actgaactgg gccacttacc ttgcaagcac agaaaacatt


 


2281
atagtagcta gctttgatgg cagaggaagt ggttaccaag gagataagat catgcatgca


 


2341
atcaacagaa gactgggaac atttgaagtt gaagatcaaa ttgaagcagc cagacaattt


 


2401
tcaaaaatgg gatttgtgga caacaaacga attgcaattt ggggctggtc atatggaggg


 


2461
tacgtaacct caatggtcct gggatcggga agtggcgtgt tcaagtgtgg aatagccgtg


 


2521
gcgcctgtat cccggtggga gtactatgac tcagtgtaca cagaacgtta catgggtctc


 


2581
ccaactccag aagacaacct tgaccattac agaaattcaa cagtcatgag cagagctgaa


 


2641
aattttaaac aagttgagta cctccttatt catggaacag cagatgataa cgttcacttt


 


2701
cagcagtcag ctcagatctc caaagccctg gtcgatgttg gagtggattt ccaggcaatg


 


2761
tggtatactg atgaagacca tggaatagct agcagcacag cacaccaaca tatatatacc


 


2821
cacatgagcc acttcataaa acaatgtttc tctttacctt agcacctcaa aataccatgc


 


2881
catttaaagc ttattaaaac tcatttttgt tttcattatc tcaaaactgc actgtcaaga


 


2941
tgatgatgat ctttaaaata cacactcaaa tcaagaaact taaggttacc tttgttccca


 


3001
aatttcatac ctatcatctt aagtagggac ttctgtcttc acaacagatt attaccttac


 


3061
agaagtttga attatccggt cgggttttat tgtttaaaat catttctgca tcagctgctg


 


3121
aaacaacaaa taggaattgt ttttatggag gctttgcata gattccctga gcaggatttt


 


3181
aatctttttc taactggact ggttcaaatg ttgttctctt ctttaaaggg atggcaagat


 


3241
gtgggcagtg atgtcactag ggcagggaca ggataagagg gattagggag agaagatagc


 


3301
agggcatggc tgggaaccca agtccaagca taccaacacg agcaggctac tgtcagctcc


 


3361
cctcggagaa gagctgttca cagccagact ggcacagttt tctgagaaag actattcaaa


 


3421
cagtctcagg aaatcaaata tgcaaagcac tgacttctaa gtaaaaccac agcagttgaa


 


3481
aagactccaa agaaatgtaa gggaaactgc cagcaacgca ggcccccagg tgccagttat


 


3541
ggctataggt gctacaaaaa cacagcaagg gtgatgggaa agcattgtaa atgtgctttt


 


3601
aaaaaaaaat actgatgttc ctagtgaaag aggcagcttg aaactgagat gtgaacacat


 


3661
cagcttgccc tgttaaaaga tgaaaatatt tgtatcacaa atcttaactt gaaggagtcc


 


3721
ttgcatcaat ttttcttatt tcatttcttt gagtgtctta attaaaagaa tattttaact


 


3781
tccttggact cattttaaaa aatggaacat aaaatacaat gttatgtatt attattccca


 


3841
ttctacatac tatggaattt ctcccagtca tttaataaat gtgccttcat tttttcagaa


 


3901
aaaaaaaaaa aaa






The atg start and stop codons are bolded and underlined. The amino acid sequence of human DPP-4 (dipeptidyl peptidase 4), provided by Genbank Accession No. NP_001926.2, is incorporated herein by reference, and is shown below (SEQ ID NO: 216).









1
mktpwkvllg llgaaalvti itvpvvllnk gtddatadsr ktytltdylk ntyrlklysl



 


61
rwisdheyly kqennilvfn aeygnssvfl enstfdefgh sindysispd gqfilleyny


 


121
vkqwrhsyta sydiydlnkr qliteeripn ntqwvtwspv ghklayvwnn diyvkiepnl


 


181
psyritwtgk ediiyngitd wvyeeevfsa ysalwwspng tflayaqfnd tevplieysf


 


241
ysdeslqypk tvrvpypkag avnptvkffv vntdslssvt natsiqitap asmligdhyl


 


301
cdvtwatqer islqwlrriq nysvmdicdy dessgrwncl varqhiemst tgwvgrfrps


 


361
ephftldgns fykiisneeg yrhicyfqid kkdctfitkg twevigieal tsdylyyisn


 


421
eykgmpggrn lykiqlsdyt kvtclsceln percqyysvs fskeakyyql rcsgpglply


 


481
tlhssvndkg lrvlednsal dkmlqnvqmp skkldfiiln etkfwyqmil pphfdkskky


 


541
pllldvyagp csqkadtvfr lnwatylast eniivasfdg rgsgyqgdki mhainrrlgt


 


601
fevedqieaa rqfskmgfvd nkriaiwgws yggyvtsmvl gsgsgvfkcg iavapvsrwe


 


661
yydsvytery mglptpednl dhyrnstvms raenfkqvey llihgtaddn vhfqqsaqis


 


721
kalvdvgvdf qamwytdedh giasstahqh iythmshfik qcfslp






The mRNA sequence encoding human CD26 provided by Genbank Accession No. M74777.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 217).









1
gacgccgacg atgaagacac cgtggaaggt tcttctggga ctgctgggtg ctgctgcgct



 


61
tgtcaccatc atcaccgtgc ccgtggttct gctgaacaaa ggcacagatg atgctacagc


 


121
tgacagtcgc aaaacttaca ctctaactga ttacttaaaa aatacttata gactgaagtt


 


181
atactcctta agatggattt cagatcatga atatctctac aaacaagaaa ataatatctt


 


241
ggtattcaat gctgaatatg gaaacagctc agttttcttg gagaacagta catttgatga


 


301
gtttggacat tctatcaatg attattcaat atctcctgat gggcagttta ttctcttaga


 


361
atacaactac gtgaagcaat ggaggcattc ctacacagct tcatatgaca tttatgattt


 


421
aaataaaagg cagctgatta cagaagagag gattccaaac aacacacagt gggtcacatg


 


481
gtcaccagtg ggtcataaat tggcatatgt ttggaacaat gacatttatg ttaaaattga


 


541
accaaattta ccaagttaca gaatcacatg gacggggaaa gaagatataa tatataatgg


 


601
aataactgac tgggtttatg aagaggaagt cttcagtgcc tactctgctc tgtggtggtc


 


661
tccaaacggc acttttttag catatgccca atttaacgac acagaagtcc cacttattga


 


721
atactccttc tactctgatg agtcactgca gtacccaaag actgtacggg ttccatatcc


 


781
aaaggcagga gctgtgaatc caactgtaaa gttctttgtt gtaaatacag actctctcag


 


841
ctcagtcacc aatgcaactt ccatacaaat cactgctcct gcttctatgt tgatagggga


 


901
tcactacttg tgtgatgtga catgggcaac acaagaaaga atttctttgc agtggctcag


 


961
gaggattcag aactattcgg tcatggatat ttgtgactat gatgaatcca gtggaagatg


 


1021
gaactgctta gtggcacggc aacacattga aatgagtact actggctggg ttggaagatt


 


1081
taggccttca gaacctcatt ttacccttga tggtaatagc ttctacaaga tcatcagcaa


 


1141
tgaagaaggt tacagacaca tttgctattt ccaaatagat aaaaaagact gcacatttat


 


1201
tacaaaaggc acctgggaag tcatcgggat agaagctcta accagtgatt atctatacta


 


1261
cattagtaat gaatataaag gaatgccagg aggaaggaat ctttataaaa tccaacttag


 


1321
tgactataca aaagtgacat gcctcagttg tgagctgaat ccggaaaggt gtcagtacta


 


1381
ttctgtgtca ttcagtaaag aggcgaagta ttatcagctg agatgttccg gtcctggtct


 


1441
gcccctctat actctacaca gcagcgtgaa tgataaaggg ctgagagtcc tggaagacaa


 


1501
ttcagctttg gataaaatgc tgcagaatgt ccagatgccc tccaaaaaac tggacttcat


 


1561
tattttgaat gaaacaaaat tttggtatca gatgatcttg cctcctcatt ttgataaatc


 


1621
caagaaatat cctctactat tagatgtgta tgcaggccca tgtagtcaaa aagcagacac


 


1681
tgtcttcaga ctgaactggg ccacttacct tgcaagcaca gaaaacatta tagtagctag


 


1741
ctttgatggc agaggaagtg gttaccaagg agataagatc atgcatgcaa tcaacagaag


 


1801
actgggaaca tttgaagttg aagatcaaat tgaagcagcc agacaatttt caaaaatggg


 


1861
atttgtggac aacaaacgaa ttgcaatttg gggctggtca tatggagggt acgtaacctc


 


1921
aatggtcctg ggatcaggaa gtggcgtgtt caagtgtgga atagccgtgg cgcctgtatc


 


1981
ccggtgggag tactatgact cagtgtacac agaacgttac atgggtctcc caactccaga


 


2041
agacaacctt gaccattaca gaaattcaac agtcatgagc agagctgaaa attttaaaca


 


2101
agttgagtac ctccttattc atggaacagc agatgataac gttcactttc agcagtcagc


 


2161
tcagatctcc aaagccctgg tcgatgttgg agtggatttc caggcaatgt ggtatactga


 


2221
tgaagaccat ggaatagcta gcagcacagc acaccaacat atatataccc acatgagcca


 


2281
cttcataaaa caatgtttct ctttacctta gcacctcaaa ataccatgcc atttaaagct


 


2341
tattaaaact catttttgtt ttcattatct caaaactgca ctgtcaagat gatgatgatc


 


2401
tttaaaatac acactcaaat caagaaactt aaggttacct ttgttcccaa atttcatacc


 


2461
tatcatctta agtagggact tctgtcttca caacagatta ttaccttaca gaagtttgaa


 


2521
ttatccggtc gggttttatt gtttaaaatc atttctgcat cagctgctga aacaacaaat


 


2581
aggaattgtt tttatggagg ctttgcatag attccctgag caggatttta atctttttct


 


2641
aactggactg gttcaaatgt tgttctcttc tttaaaggga tggcaagatg tgggcagtga


 


2701
tgtcactagg gcagggacag gataagaggg attagggaga gaagatagca gggcatggct


 


2761
gggaacccaa gtccaagcat accaacacga ccaggctact gtcagctccc ctcggagaaa


 


2821
actgtgcagt ctgcgtgtga acagctcttc tcctttagag cacaatggat ctcgagggat


 


2881
cttccatacc taccagttct gcgcctcgag gccgcgactc taga






The atg start and stop codons are bolded and underlined. The amino acid sequence of human CD26, provided by Genbank Accession No. AAA51943.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 218).









1
mktpwkvllg llgaaalvti itvpvvllnk gtddatadsr ktytltdylk ntyrlklysl



 


61
rwisdheyly kqennilvfn aeygnssvfl enstfdefgh sindysispd gqfilleyny


 


121
vkqwrhsyta sydiydlnkr qliteeripn ntqwvtwspv ghklayvwnn diyvkiepnl


 


181
psyritwtgk ediiyngitd wvyeeevfsa ysalwwspng tflayaqfnd tevplieysf


 


241
ysdeslqypk tvrvpypkag avnptvkffv vntdslssvt natsiqitap asmligdhyl


 


301
cdvtwatqer islqwlrriq nysvmdicdy dessgrwncl varqhiemst tgwvgrfrps


 


361
ephftldgns fykiisneeg yrhicyfqid kkdctfitkg twevigieal tsdylyyisn


 


421
eykgmpggrn lykiqlsdyt kvtclsceln percqyysvs fskeakyyql rcsgpglply


 


481
tlhssvndkg lrvlednsal dkmlqnvqmp skkldfiiln etkfwyqmil pphfdkskky


 


541
pllldvyagp csqkadtvfr lnwatylast eniivasfdg rgsgyqgdki mhainrrlgt


 


601
fevedqieaa rqfskmgfvd nkriaiwgws yggyvtsmvl gsgsgvfkcg iavapvsrwe


 


661
yydsvytery mglptpednl dhyrnstvms raenfkqvey llihgtaddn vhfqqsaqis


 


721
kalvdvgvdf qamwytdedh giasstahqh iythmshfik qcfslp






The mRNA sequence encoding human SIRT1 provided by Genbank Accession No. JQ768366.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 219).









1
atgattggca cagatcctcg aacaattctt aaagatttat tgccggaaac aatacctcca



 


61
cctgagttgg atgatatgac actgtggcag attgttatta atatcctttc agaaccacca


 


121
aaaaggaaaa aaagaaaaga tattaataca attgaagatg ctgtgaaatt actgcaagag


 


181
tgcaaaaaaa ttatagttct aactggagct ggggtgtctg tttcatgtgg aatacctgac


 


241
ttcaggtcaa gggatggtat ttatgctcgc cttgctgtag acttcccaga tcttccagat


 


301
cctcaagcga tgtttgatat tgaatatttc agaaaagatc caagaccatt cttcaagttt


 


361
gcaaaggaaa tatatcctgg acaattccag ccatctctct gtcacaaatt catagccttg


 


421
tcagataagg aaggaaaact acttcgcaac tatacccaga acatagacac gctggaacag


 


481
gttgcgggaa tccaaaggat aattcagtgt catggttcct ttgcaacagc atcttgcctg


 


541
atttgtaaat acaaagttga ctgtgaagct gtacgaggag ctctttttag tcaggtagtt


 


601
cctcgatgtc ctaggtgccc agctgatgaa ccgcttgcta tcatgaaacc agagattgtg


 


661
ttttttggtg aaaatttacc agaacagttt catagagcca tgaagtatga caaagatgaa


 


721
gttgacctcc tcattgttat tgggtcttcc ctcaaagtaa gaccagtagc actaattcca


 


781
agttccatac cccatgaagt gcctcagata ttaattaata gagaaccttt gcctcatctg


 


841
cattttgatg tagagcttct tggagactgt gatgtcataa ttaatgaatt gtgtcatagg


 


901
ttaggtggtg aatatgccaa actttgctgt aaccctgtaa agctttcaga aattactgaa


 


961
aaacctccac gaacacaaaa agaattggct tatttgtcag agttgccacc cacacctctt


 


1021
catgtttcag aagactcaag ttcaccagaa agaacttcac caccagattc ttcagtgatt


 


1081
gtcacacttt tagaccaagc agctaagagt aatgatgatt tagatgtgtc tgaatcaaaa


 


1141
ggttgtatgg aagaaaaacc acaggaagta caaacttcta ggaatgttga aagtattgct


 


1201
gaacagatgg aaaatccgga tttgaagaat gttggttcta gtactgggga gaaaaatgaa






The atg start and stop codons are bolded and underlined. The amino acid sequence of human SIRT1, provided by Genbank Accession No. JQ768366.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 220).









1
migtdprtil kdllpetipp pelddmtlwq ivinilsepp krkkrkdint iedavkllqe



 


61
ckkiivltga gvsvscgipd frsrdgiyar lavdfpdlpd pqamfdieyf rkdprpffkf


 


121
akeiypgqfq pslchkfial sdkegkllrn ytqnidtleq vagiqriiqc hgsfatascl


 


181
ickykvdcea vrgalfsqvv prcprcpade plaimkpeiv ffgenlpeqf hramkydkde


 


241
vdllivigss lkvrpvalip ssiphevpqi linreplphl hfdvellgdc dviinelchr


 


301
lggeyaklcc npvklseite kpprtqkela ylselpptpl hvsedssspe rtsppdssvi


 


361
vtlldqaaks nddldvsesk gcmeekpqev qtsrnvesia eqmenpdlkn vgsstgekne






The mRNA sequence encoding human FoxO3a (forkhead box O3) provided by Genbank Accession No. NM_001455.3, is incorporated herein by reference, and is shown below (SEQ ID NO: 221).









1
gcgcgaggcc gtcgattcgc tcgcggctcc atcgcggcct ggccgggggg cggtgtctgc



 


61
tgcgccaggt tcgctggccg cacgtcttca ggtcctcctg ttcctgggag gcgggcgcgg


 


121
caggactggg aggtggcggc agcgggcgag gactcgccga ggacggggct ccggcccggg


 


181
ataaccaact ctccttctct cttctttggt gcttccccag gcggcggcgg cggcgcccgg


 


241
gagccggagc cttcgcggcg tccacgtccc tcccccgctg caccccgccc cggcgcgaga


 


301
ggagagcgcg agagccccag ccgcgggcgg gcgggcggcg aagatggcag aggcaccggc


 


361
ttccccggcc ccgctctctc cgctcgaagt ggagctggac ccggagttcg agccccagag


 


421
ccgtccgcga tcctgtacgt ggcccctgca aaggccggag ctccaagcga gccctgccaa


 


481
gccctcgggg gagacggccg ccgactccat gatccccgag gaggaggacg atgaagacga


 


541
cgaggacggc gggggacggg ccggctcggc catggcgatc ggcggcggcg gcgggagcgg


 


601
cacgctgggc tccgggctgc tccttgagga ctcggcccgg gtgctggcac ccggagggca


 


661
agaccccggg tctgggccag ccaccgcggc gggcgggctg agcgggggta cacaggcgct


 


721
gctgcagcct cagcaaccgc tgccaccgcc gcagccgggg gcggctgggg gctccgggca


 


781
gccgaggaaa tgttcgtcgc ggcggaacgc ctggggaaac ctgtcctacg cggacctgat


 


841
cacccgcgcc atcgagagct ccccggacaa acggctcact ctgtcccaga tctacgagtg


 


901
gatggtgcgt tgcgtgccct acttcaagga taagggcgac agcaacagct ctgccggctg


 


961
gaagaactcc atccggcaca acctgtcact gcatagtcga ttcatgcggg tccagaatga


 


1021
gggaactggc aagagctctt ggtggatcat caaccctgat ggggggaaga gcggaaaagc


 


1081
cccccggcgg cgggctgtct ccatggacaa tagcaacaag tataccaaga gccgtggccg


 


1141
cgcagccaag aagaaggcag ccctgcagac agcccccgaa tcagctgacg acagtccctc


 


1201
ccagctctcc aagtggcctg gcagccccac gtcacgcagc agtgatgagc tggatgcgtg


 


1261
gacggacttc cgttcacgca ccaattctaa cgccagcaca gtcagtggcc gcctgtcgcc


 


1321
catcatggca agcacagagt tggatgaagt ccaggacgat gatgcgcctc tctcgcccat


 


1381
gctctacagc agctcagcca gcctgtcacc ttcagtaagc aagccgtgca cggtggaact


 


1441
gccacggctg actgatatgg caggcaccat gaatctgaat gatgggctga ctgaaaacct


 


1501
catggacgac ctgctggata acatcacgct cccgccatcc cagccatcgc ccactggggg


 


1561
actcatgcag cggagctcta gcttcccgta taccaccaag ggctcgggcc tgggctcccc


 


1621
aaccagctcc tttaacagca cggtgttcgg accttcatct ctgaactccc tacgccagtc


 


1681
tcccatgcag accatccaag agaacaagcc agctaccttc tcttccatgt cacactatgg


 


1741
taaccagaca ctccaggacc tgctcacttc ggactcactt agccacagcg atgtcatgat


 


1801
gacacagtcg gaccccttga tgtctcaggc cagcaccgct gtgtctgccc agaattcccg


 


1861
ccggaacgtg atgcttcgca atgatccgat gatgtccttt gctgcccagc ctaaccaggg


 


1921
aagtttggtc aatcagaact tgctccacca ccagcaccaa acccagggcg ctcttggtgg


 


1981
cagccgtgcc ttgtcgaatt ctgtcagcaa catgggcttg agtgagtcca gcagccttgg


 


2041
gtcagccaaa caccagcagc agtctcctgt cagccagtct atgcaaaccc tctcggactc


 


2101
tctctcaggc tcctccttgt actcaactag tgcaaacctg cccgtcatgg gccatgagaa


 


2161
gttccccagc gacttggacc tggacatgtt caatgggagc ttggaatgtg acatggagtc


 


2221
cattatccgt agtgaactca tggatgctga tgggttggat tttaactttg attccctcat


 


2281
ctccacacag aatgttgttg gtttgaacgt ggggaacttc actggtgcta agcaggcctc


 


2341
atctcagagc tgggtgccag gctgaaggat cactgaggaa ggggaagtgg gcaaagcaga


 


2401
ccctcaaact gacacaagac ctacagagaa aaccctttgc caaatctgct ctcagcaagt


 


2461
ggacagtgat accgtttaca gcttaacacc tttgtgaatc ccacgccatt ttcctaaccc


 


2521
agcagagact gttaatggcc ccttaccctg ggtgaagcac ttacccttgg aacagaactc


 


2581
taaaaagtat gcaaaatctt ccttgtacag ggtggtgagc cgcctgccag tggaggacag


 


2641
cacccctcag caccacccac cctcattcag agcacaccgt gagcccccgt cggccattct


 


2701
gtggtgtttt aatattgcga tggtttatgg gacgttttaa gtgttgttct tgtgtttgtt


 


2761
ttcctttgac tttctgagtt tttcacatgc attaacttgc ggtatttttc tgttaaaatg


 


2821
ttaaccgtcc ttcccctagc aaatttaaaa acagaaagaa aatgttgtac cagttaccat


 


2881
tccgggttcg agcatcacaa gcttttgagc gcatggaact ccataaacta acaaattaca


 


2941
taaactaaag ggggattttc tttcttcttt tgtttggtag aaaattatcc ttttctaaaa


 


3001
actgaacaat ggcacaattg tttgctatgt gcacccgtcc aggacagaac cgtgcatagg


 


3061
caaaaggagt ggagcacagc gtccggccca gtgtgtttcc ggttctgagt cagggtgatc


 


3121
tgtggacggg accccagcac caagtctacg ggtgccagat cagtagggcc tgtgatttcc


 


3181
tgtcagtgtc ctcagctaat gtgaacagtg ttggtctgct ggttagaaac tagaatattg


 


3241
atattttcag gaaagaaatc agctcagctc tccactcatt gccaaatgtc actaaagggt


 


3301
ttagttttaa ggagaaagaa aaggaaaaaa aaaaaaaaca aaaaagtcct gttttgcttt


 


3361
gcagaacaaa tgaacttaca ggtgagcatt aagcttgcag tgagaaatgt gcgaagagta


 


3421
aaaacccaag tcaatgctga ggcagttcta acttcactgt tttcctaaat acacatcctt


 


3481
gattattttc agccttgcta tataatctga tctgctagaa gtgtatgagt gagaggcaat


 


3541
agcatacaaa ctgatttttt aaatataagc ttaggttgta attgtacaag tgactcaatg


 


3601
gaagtacaaa atagggcagt tttaactttt ttttctgctt ctatggattt cattttgttg


 


3661
tgttttcaaa aagttatggt gctgtatagg tgctttctgt ttaacctgga aagtgtgatt


 


3721
atattcgtta ccttctttgg tagacggaat agttgggacc acctttggta cataagaaat


 


3781
tggtataacg atgctctgat tagcacagta tatgcatact tctccaaagt gatatatgaa


 


3841
gactcttttc tttgcataaa aagcattagg catataaatg tataaatata ttttatcatg


 


3901
tacagtacaa aaatggaacc ttatgcatgg gccttaggaa tacaggctag tatttcagca


 


3961
cagacttccc tgcttgagtt cttgctgatg cttgcaccgt gacagtgggc accaacacag


 


4021
acgtgccacc caaccccctg cacacaccac cggccaccag gggccccctt gtgcgccttg


 


4081
gctttataac tcctctgggg gtgatattgg tggtgatcac agctcctagc ataatgagag


 


4141
ttccatttgg tattgtcaca cgtctcctgc ctcgcttggg ttgccatgtt tgagcgatgg


 


4201
ccctgttgat ttcaccctgc cttttactga atctgtaaat tgttgtgcaa ttgtggttat


 


4261
agtagactgt agcacattgc cttttctaaa ctgctacatg tttataatct tcatttttaa


 


4321
agtatgtgta atttttttaa gtatgtattc tattcatatg gtctgcttgt cagtgagcca


 


4381
gacttgctta ctatattcct ttataataat gctagccact tcctggattc tttagtaatg


 


4441
tgctgtatgc aagaactttc cagtagcagt gaaggagggt tgcctctcca agcttcctaa


 


4501
gggatgctgc cctgtgtggg gatgcattgc agaggcacta gtagcatggg ggctagagtg


 


4561
gggagcgaga tgtaaaaggg tggggggata ggagaattcc agagtgcttc cagcattagg


 


4621
gtcctgagaa cttctgagtt cagagaaaca tgcaaagtga ctaacaaaat agctacttac


 


4681
ctttgcagtt ttacagaccc tgggagctgc tttgggagtg agaaaggcaa ccctccaatg


 


4741
tgtttcaact ttaaaatgtt gaattctttt cagacatggt atctcattta ttctcctttt


 


4801
ctagcgtttg ttgaatttca ggcagaatgt cttacagaat gtcctagaac cagattatca


 


4861
tttaatctga aacagctgag gaagggacag agaaggtaca agggcaaggc agcacaaaac


 


4921
agatcaggag aatgaagagg gaatgctttg gttttttgtt ttgttttgtt ttttcttttt


 


4981
caagtaacta aaacagcatc tacatgtaga gtgttgtgga gagctgagac cagggtaaag


 


5041
tcaagtgcag catcagtact gcgagaccca ccagcccctg gagagggtca gccgagaatc


 


5101
tggtagtgaa gcctgtctag ggtcccggca ccctcaccct cagccacctg cagagaggcc


 


5161
agggccccag agactagcct ggttctgaag tgggcagggg tgctgccaga gccctctgcc


 


5221
ccttatgttg agaccctgct ttcaggacag gccagccgtt ggccaccatg tcacattctg


 


5281
agtgagtgtc acaggtccct aacaataatt ttctgatctg gagcatatca gcagaatgct


 


5341
tagcctcaag gggcctggca gctgtaatgt ttgatttatg atgagaacta tccgaggcca


 


5401
cccttggcct ctaaataagc tgctctaggg agccgcctac tttttgatga gaaattagaa


 


5461
gagtacctaa tgttgaaaac atgacatgcg ctcttgggat ctgctgttct ctccagggct


 


5521
ccagaacctg atacctgtta ccaaagctag gaaagagctt tatcacaagc cttcactgtc


 


5581
ctggcatgag aactggctgc caggctcagt gtaccccatt aactgtgaat gaatctgagc


 


5641
ttggtttcct ttattgcttc ctctgcaata tgattgctga aacacatttt aaaaattcag


 


5701
aagcttgtca ctcctgttaa tgggaggatc agtcacacat gtgtagtaca aggcggactt


 


5761
tgtgtttgtt tttggtgtta atttttagca ttgtgtgtgt tgcttcccca ccctgaggag


 


5821
aggacaccat ggcttactac tcaggacaag tatgccccgc tcagggtgtg atttcaggtg


 


5881
gcttccaaac ttgtacgcag tttaaagatg gtggggacag actttgcctc tacctagtga


 


5941
accccactta aagaataagg agcatttgaa tctcttggaa aaggccatga agaataaagc


 


6001
agtcaaaaag aagtcctcca tgttggtgcc aaggacttgc gaggggaaat aaaaatgtta


 


6061
tccagcctga ccaacatgga gaaaccccgt ctccattaaa aatacaaaat tagcctggca


 


6121
tggtggcgca tgcctgtaat cccagctact ctggaggctg aggcaggaga atcgcttgaa


 


6181
cccaggaggc ggaggtcgca gtgagccgag atcatgccag tgcactccag cctgggtaac


 


6241
aagagtgaaa ctccgtgtca aaaaaaaaaa aaaaatgtta ctcatcctct ctgaaagcaa


 


6301
aaaggaaacc ctaacagctc tgaactctgg ttttattttt cttgctgtat ttgggtgaac


 


6361
attgtatgat taggcataat gttaaaaaaa aaaatttttt tttggtagaa atgcaatcac


 


6421
cagtaaagag gtacgaaaaa gctagcctct ctcagagacc ggggaggcag agtactacta


 


6481
gaggaagtga agttctgatg gaatcatgcc tgtcaaatga ggtcttgaag cggatgccca


 


6541
aataaaagag tatattttat ctaaatctta agtgggtaac attttatgca gtttaaatga


 


6601
atggaatatt ttcctcttgt ttagttgtat ctgtttgtat ttttctttga tgaatgattg


 


6661
gtcatgaggc ctcttgccac actccagaaa tacgtgtgcg gctgctttta agaactatgt


 


6721
gtctggtcac ttatttctct aaaattatct cattgcctgg caatcagtct tctcttgtat


 


6781
acttgtccta gcacattatg tacatgggaa atgtaaacaa atgtgaagga ggaccagaaa


 


6841
aattagttaa tatttaaaaa aatgtattgt gcattttggc ttcacatgtt taactttttt


 


6901
taagaaaaaa gttgcatgaa tggaaaaaaa aatctgtata cagtatctgt aaaaactatc


 


6961
ttatctgttt caattccttg ctcatatccc atataatcta gaactaaata tggtgtgtgg


 


7021
ccatatttaa acacctgaga gtcaagcagt tgagactttg atttgaagca cctcatcctt


 


7081
ctttcaatgc gaacactatc atatggcatt cttactgagg attttgtcta accatatgtt


 


7141
gccatgaatt aactctgccg cctttcttaa ggatcaaaac cagtttgatt tgggaatctt


 


7201
cccctttcca aatgaaatag agatgcagta cttaactttc cttggtgttt gtagatattg


 


7261
ccttgtgtat tccacttaaa accgtaatct agtttgtaaa agagatggtg acgcatgtaa


 


7321
ataaagcatc agtgacactc t






The atg start and stop codons are bolded and underlined. The amino acid sequence of human FoxO3a (forkhead box O3), provided by Genbank Accession No. NP_001446.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 222).








(SEQ ID NO: 222)










1
maeapaspap lspleveldp efepqsrprs ctwplqrpel qaspakpsge taadsmipee



 


61
eddeddedgg gragsamaig ggggsgtlgs gllledsarv lapggqdpgs gpataaggls


 


121
ggtqallqpq qplpppqpga aggsgqprkc ssrrnawgnl syadlitrai esspdkrltl


 


181
sqiyewmvrc vpyfkdkgds nssagwknsi rhnlslhsrf mrvqnegtgk sswwiinpdg


 


241
gksgkaprrr avsmdnsnky tksrgraakk kaalqtapes addspsqlsk wpgsptsrss


 


301
deldawtdfr srtnsnastv sgrlspimas teldevqddd aplspmlyss saslspsvsk


 


361
pctvelprlt dmagtmnlnd gltenlmddl ldnitlppsq psptgglmqr sssfpyttkg


 


421
sglgsptssf nstvfgpssl nslrqspmqt iqenkpatfs smshygnqtl qdlltsdsls


 


481
hsdvmmtqsd plmsqastav saqnsrrnvm lrndpmmsfa aqpnqgslvn qnllhhqhqt


 


541
qgalggsral snsvsnmgls essslgsakh qqqspvsqsm qtlsdslsgs slystsanlp


 


601
vmghekfpsd ldldmfngsl ecdmesiirs elmdadgldf nfdslistqn vvglnvgnft


 


661
gakqassqsw vpg






The mRNA sequence encoding human MiR-24 provided by Genbank Accession No. AF480527.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 223).









1
tggctcagtt cagcaggaac ag
(SEQ ID NO: 223)






The mRNA sequence encoding human MiR-125a-5p (hsa-mir-125a) provided by Genbank Accession No. LM608509.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 224).









1
tgccagtctc taggtccctg agacccttta acctgtgagg acatccaggg tcacaggtga



 


61
ggttcttggg agcctggcgt ctggcc






The mRNA sequence encoding human MiR-203a (MiR-203), provided by Genbank Accession No. NR_029620.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 225).









1
gtgttgggga ctcgcgcgct gggtccagtg gttcttaaca gttcaacagt tctgtagcgc



 


61
aattgtgaaa tgtttaggac cactagaccc ggcgggcgcg gcgacagcga






The mRNA sequence encoding human MiR-140, provided by Genbank Accession No. NR_029681.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 226).









1
tgtgtctctc tctgtgtcct gccagtggtt ttaccctatg gtaggttacg tcatgctgtt



 


61
ctaccacagg gtagaaccac ggacaggata ccggggcacc






The mRNA sequence encoding human MiR-27a, provided by Genbank Accession No. NR_029501.1, is incorporated herein by reference, and is shown below (SEQ ID NO: 227).









1
ctgaggagca gggcttagct gcttgtgagc agggtccaca ccaagtcgtg ttcacagtgg



 


61
ctaagttccg ccccccag






Formulation and Dosing

In accordance with certain examples, a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Such pharmaceutical compositions may be administered by injection or infusion into a localized tissue site, e.g., into an articulating joint or by inhalation, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, intra-articularly, subcutaneously, intravenously or other suitable methods that will be readily selected by the person of ordinary skill in the art, given the benefit of this disclosure. For example, solutions or suspensions used for parenteral, intradermal, intra-articular, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral and/or intra-articular preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
A biologically acceptable medium includes, but is not limited to, any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the complexes of the present disclosure. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the activity of the small molecule, protein, polypeptide and/or peptide, its use in the pharmaceutical preparation of the invention is contemplated. Suitable vehicles and formulations are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable formulations.
The complexes of the present invention may be administered by any suitable route. For example, a pharmaceutical preparation may be administered in tablets or capsules, by injection, by infusion, by inhalation, topically (e.g., by lotion or ointment), by suppository, by controlled release patch, or the like.
The complexes described herein may be administered to an individual (e.g., a human or animal such as a non-human primate) for therapy by any suitable route of administration, including orally, nasally, rectally, intravaginally, parenterally, intra-articularly, intracisternally, topically, buccally, sublingually, epidurally and the like. Intra-articular administration is useful for local treatment of disease and flare-up, e.g. pain in joints, synovitis and the like.
Regardless of the route of administration selected, the pharmaceutical compositions of the present invention are formulated into pharmaceutically acceptable dosage forms such as described below or by other conventional methods known to those of skill in the art. Actual dosage levels of the pharmaceutical compositions described herein may be varied so as to obtain an amount of the compound which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
Joint disease is treated using the complexes or compositions described herein. For example, methods are provided for treating a patient having a joint disease, by administering to the patient a therapeutically effective amount of a complex or composition of the present invention. For in vivo therapies based on local injection (e.g., intratumoral, intraarticularly, intramuscularly, into the peritoneal cavity, and aerosolized treatments) the RNT/small RNA complex is advantageously water soluble and so may be administered as an aqueous injection.
The selected dosage level will depend upon a variety of factors including the activity of a particular compound or ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular complex employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician, veterinarian or research scientist having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician, veterinarian or research scientist could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Furthermore, different delivery materials are used to administer different doses and dose ranges. For example, Nanopieces demonstrate good biocompatibility and low toxicity. Previous studies have demonstrated no significant toxicity with an administration of 25 μg delivery nanotubes (RNTs) in vivo (Journeay W S, et al. Int J Nanomedicine. 2008; 3(3):373-83). Even with a 50 μg dose, inflammation that resulted from RNTs was resolved after 7 days. In comparison, some conventional delivery materials such as carbon nanotubes, can cause inflammation at much lower doses the resulting in inflammation that can last for two months. In the current system, a 5 μg dose of RNT in Nanopiece was effective in the delivery of cargo. Therefore, the effective doses of RNT Nanopieces are significantly lower than their toxic doses, providing a good therapeutic index. Moreover, RNTs or TBLs showed a lower toxicity than lipid-based delivery vehicles. In FIG. 66, ATDC5 cells were cultured with no additives (negative control), Nanopieces of 0.1 nmol non-targeting siRNA with 10 μg of RNT, Nanopieces of 0.1 nmol non-targeting siRNA with 2.5 μg TBL, or 0.1 nmol non-targeting siRNA with 6 μg Lipofectamine 2000. After 24 hours, ATDC5 cells cultured with Lipofectamine 2000 showed abnormal cell morphology and large amount of cell debris, however, cells cultured with either RNT nanopiece or TBL nanopiece presented normal morphology as the negative control.
In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, doses of the compounds of this invention for a patient will range from about 0.0001 to about 100 mg per kilogram of body weight per day, or from about 0.001 to 30 mg/kg body weight, from about 0.01 to 25 mg/kg body weight, from about 0.1 to 20 mg/kg body weight, from about 1 to 10 mg/kg, from about 2 to 9 mg/kg, from about 3 to 8 mg/kg, from about 4 to 7 mg/kg, or from about 5 to 6 mg/kg body weight.
The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of biologically active agent can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result from the results of diagnostic assays.
If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments, an effective dose is given every other day, twice a week, once a week or once a month.
A complex of the invention can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with other antimicrobial agents such as penicillin, cephalosporin, aminoglycosides, glycopeptides and the like. Conjunctive therapy includes sequential, simultaneous and separate administration of an active compound in such a way that the therapeutic effects of the first administered compound are still present when a subsequent administration is performed.
Another aspect of the present disclosure provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of the complexes described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present disclosure may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection or intraarticularly as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam. However, in certain embodiments the subject complexes may be simply dissolved or suspended in sterile water.
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in compositions of the present invention.
Examples of pharmaceutically acceptable antioxidants include but are not limited to: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Formulations of the present invention may conveniently be presented in unit dosage form and may be prepared by any methods well known in the pharmaceutical art. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the individual being treated and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, from about 5 percent to about 70 percent, from about 10 percent to about 30 percent, from about 15 percent to about 25 percent, or from about 18 percent to about 22 percent. In an alternative embodiment, compounds of the present invention can be administered per se, e.g., in the absence of carrier material.
Methods of preparing the formulations or compositions of the present invention include the step of associating a complex described herein with a carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly associating a complex of the present invention with liquid carriers, finely divided solid carriers, or both, and, optionally, shaping the product.
Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, such as sucrose and acacia or tragacanth), powders, granules, as a solution or a suspension in an aqueous or non-aqueous liquid, as an oil-in-water or water-in-oil liquid emulsion, as an elixir or syrup, as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a complex of the present invention as an active ingredient. A complex of the present invention may also be administered as a bolus, electuary or paste.
Ointments, pastes, creams and gels may contain, in addition to a complex of the present disclosure, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to a complex of the present disclosure, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
Transdermal patches have the added advantage of providing controlled delivery of a complex of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the complex in the proper medium. Absorption enhancers can also be used to increase the flux of the complex across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the complex in a polymer matrix or gel.
Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more complexes of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol asorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
Injectable depot forms are made by forming microencapsule matrices of the complexes in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
In accordance with certain examples, complexes of the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the complexes disclosed here and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
In accordance with certain examples, a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Such pharmaceutical compositions may be administered by inhalation, intraarticularly, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously, intravenously or other suitable methods that will be readily selected by the person of ordinary skill in the art, given the benefit of this disclosure. For example, solutions or suspensions used for parenteral, intraarticularly, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
The present disclosure is directed to methods of forming a delivery complex, for example, by mixing one or more agents with fully formed rosette nanotubes or modules that self-assemble into rosette nanotubes, such as the compounds of Formula I or Formula II. According to one aspect, fully formed rosette nanotubes in the form of a powder is dissolved in water and heated to boiling. The solution is then cooled to room temperature. One or more agents is then added to the solution of nanotubes at a suitable temperature and for a suitable period of time until a complex of the nanotube and one or more agents forms. Suitable ratios of the nucleic acid to nanotube include about 0.01:1 (wt/wt) to about 1:0.1 (wt/wt).
Definitions
“Alkyl”, as used herein, refers to the radical of saturated or unsaturated aliphatic groups, including straight-chain alkyl, alkenyl, or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups, cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkyl substituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, and cycloalkyl substituted alkyl, alkenyl, or alkynyl groups. Unless otherwise indicated, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), more preferably 20 or fewer carbon atoms, more preferably 12 or fewer carbon atoms, and most preferably 8 or fewer carbon atoms. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. The ranges provided above are inclusive of all values between the minimum value and the maximum value.
The term “alkyl” includes both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having one or more substituents replacing hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amino, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.
Unless the number of carbons is otherwise specified, “lower alkyl” as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Preferred alkyl groups are lower alkyls.
The alkyl groups may also contain one or more heteroatoms within the carbon backbone. Preferably the heteroatoms incorporated into the carbon backbone are oxygen, nitrogen, sulfur, and combinations thereof. In certain embodiments, the alkyl group contains between one and four heteroatoms.
“Alkenyl” and “Alkynyl”, as used herein, refer to unsaturated aliphatic groups containing one or more double or triple bonds analogous in length (e.g., C2-C30) and possible substitution to the alkyl groups described above.
“Halogen”, as used herein, refers to fluorine, chlorine, bromine, or iodine.
The term “optionally substituted” as used herein, refers to all permissible substituents of the compounds described herein. In the broadest sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aryloxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups.
Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e. a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
The term “amino acid” is inclusive of the 20 common amino acids, as well as “nonstandard amino acids,” for example, D-amino acids and chemically (or biologically) produced derivatives of “common” amino acids, including for example, β-amino acids. Accordingly, amino acids according to the present disclosure include the commonly known amino acids such as glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), proline (Pro, P), hydroxyproline, phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (Trp, W) cysteine (Cys, C), methionine (Met, M) serine (Ser, S), o-phosphoserine, threonine (Thr, T), lysine (Lys, K), arginine (Arg, R), histidine (His, H), aspartate (Asp, D), glutamate (Glu, E), γ-carboxyglutamate, asparagine (Asn, N), glutamine (Gln, Q) and the like. Amino acids also include stereoisomers thereof and compounds structurally similar to the amino acids or modifications or derivatives thereof. Exemplary amino acids within the scope of the present disclosure include lysine, arginine, serine, glycine, aspartate and the like. The amino acids of the present disclosure are modified only at their terminal amine group.
Amino acids are composed of amine (—NH2) and carboxylic acid (—COOH) functional groups, along with a side-chain specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, though other elements are found in the side-chains of certain amino acids.
In the structure shown below, Z represents a side-chain specific to each amino acid. The carbon atom next to the carboxyl group (which is therefore numbered 2 in the carbon chain starting from that functional group) is called the α-carbon. Amino acids containing an amino group bonded directly to the alpha carbon are referred to as alpha amino acids.




Amino acids can be divided into amino acid containing hydrophilic side chains, hydrophobic side chains, and electrically charged side chains. See FIG. 69, wherein the side chains are shaded.

The term “peptide” is inclusive of both straight and branched amino acid chains, as well as cyclic amino acid chains, which comprise at least 2 amino acid residues. The terms “peptide” and “polypeptide” are used interchangeably herein. Accordingly, polypeptides according to the present disclosure include two or more amino acids covalently linked together. According to one aspect, the two or more amino acids are covalently linked together at least in part by one or more peptide bonds. The polypeptides of the present disclosure are modified only at their terminal amine group. For example, the peptide or fragment of a full-length protein comprises 2, 5, 10, 50, 100, 200, 500 600, 700, 750, 800, 900, 1000 or more amino acids in length or up to the full length of a reference protein.
As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer. Polynucleotides useful in the methods of the invention may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences. As used herein, one of skill in the art will understand that the term “nucleic acid probe” includes probes known as molecular beacons which include synthetic oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions or in cells. Species of molecular beacons include hairpin shaped molecules with a detectable marker such as an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid sequence. Technically, molecular beacons can be designed to target any gene and can be linked with fluorescent molecules of different fluorescence wavelengths.
A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotides.
Examples of modified nucleotides include, but are not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine and the like. Nucleic acid molecules may also be modified at the base moiety, sugar moiety or phosphate backbone.
The term “small RNA” is used as it is in the art, and includes a duplex of RNA (30 bases or less in each strand) that targets mRNA. Small RNA may be chemically or enzymatically synthesized. Small RNA in accordance with the present invention may be incorporated and then activated in RISC (RNA-induced silencing complex).
A “therapeutically effective amount” is an amount necessary to prevent, delay or reduce the severity of the onset of disease, or an amount necessary to arrest or reduce the severity of an ongoing disease, and also includes an amount necessary to enhance normal physiological functioning.
The word “transfect” is broadly used herein to refer to introduction of an exogenous compound, such as a polynucleotide sequence, into a prokaryotic or eukaryotic cell; the term includes, without limitation, introduction of an exogenous nucleic acid into a cell, which may result in a permanent or temporary alteration of genotype in an immortal or non-immortal cell line. Accordingly, embodiments of the present disclosure include the introduction of a polynucleotide sequence to either be expressed or to inhibit expression of a target gene.
As may be used herein, the terms “drug,” biologically active agent,” and “therapeutic agent” are used interchangeably and are intended to include, but are not limited to, those compounds recognized by persons of skill in the art as being biologically active agents, or drugs or therapeutic agents and include any synthetic or natural element or compound which when introduced into the body causes a desired biological response, such as altering body function.
As used herein, the terms “parenteral administration” and “administered parenterally” are intended to include, but are not limited to, modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal injection, intrasternal injection, infusion and the like.
As used herein, the terms “systemic administration,” “administered systemically,” “peripheral administration” and “administered peripherally” are intended to include, but are not limited to, the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters an individual's system and, thus, is subject to metabolism and other like processes, such as, for example, subcutaneous administration.
The term “treatment,” as used herein, is intended to include, but is not limited to, prophylaxis, therapy and cure. A patient or individual receiving treatment is any animal in need, such as humans, non-human primates, and other mammals such as horses, camels, cattle, swine, sheep, poultry, goats, rabbits, mice, guinea pigs, dogs, cats and the like.
As used herein, the term “therapeutically effective amount” is intended to include, but is not limited to, an amount of a compound, material, or composition comprising a complex of the present invention which is effective for producing a desired therapeutic effect in at least a subpopulation of cells in an animal and thereby altering (e.g., reducing or increasing) the biological consequences of one or more pathways in the treated cells, at a reasonable benefit/risk ratio.
As used herein, the term “pharmaceutically acceptable” is intended to include, but is not limited to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
As used herein, a “pharmaceutically acceptable agent” (such as a salt, carrier, excipient or diluent) is a component which (1) is compatible with the RNT/small RNA composites in that it can be included in the delivery composition without eliminating the capacity of the RNT/small RNA composites to transfect cells and deliver small RNA; and (2) where the delivery composition is intended for therapeutic uses, is suitable for use with an animal (e.g., a human) without undue adverse side effects, such as toxicity, irritation, and allergic response. Side effects are “undue” when their risk outweighs the benefit provided by the pharmaceutical agent.
As used herein, the term “pharmaceutically acceptable carrier” is intended to include, but is not limited to, a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the complexes of the present disclosure from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not unduly dangerous to the patient. Examples of materials which can serve as pharmaceutically acceptable carriers include but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations, which could easily be determined by one of skill in the art.
Chemical compounds, polynucleotides, polypeptides, and oligosaccharides of the invention are purified and/or isolated. Purified defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents. Specifically, as used herein, an “isolated” or “purified” compound, nucleic acid molecule, polynucleotide, polypeptide, protein or oligosaccharide, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. For example, purified compositions are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. For example, a purified compound refers to a one that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. Preferably, the compound constitutes at least 10, 20, 50, 70, 80, 90, 95, 99-100% by dry weight of the purified preparation.
By “isolated nucleic acid” is meant a nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule, but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner, such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones.
As used therein, the term “patient” is intended to include a mammal suffering from a disease. Such a mammal can be a human or another animal such as a companion animal (e.g., dog or cat) or a performance animal or livestock animal (e.g., an equine, bovine, porcine animal).
EXAMPLES
The following examples are specific embodiments of the present invention but are not intended to limit it.
Example 1
Nanopieces that include RNTs and exemplary cargo or payload compounds were manufactured. Cargo agents assemble with RNTs into Nanopieces. Then, taking siRNA Nanopiece as an example, it was demonstrated that Nanopieces can be intentionally processed into different sizes and charge for matrix penetration, e.g. preferential delivery of the cargo to specific tissue types. For example, Nanopieces with a net positive charge were made to deliver payload compounds to negatively charged tissue such as cartilage.
The relation between RNT/siRNA ratio and surface charge was evaluated. Selecting the ratio to result in a net positive charge on Nanopieces, Nanopieces have better binding and longer retention time on negatively charged tissue matrix (e.g., human articular cartilage).
For in vitro and in vivo delivery studies, cartilage was used as an example, because cartilage is an avascular tissue with high matrix component, which is a challenging tissue for drug delivery. Other target matrix and/or tissue can be used and the net charge of the Nanopiece tuned for preferential targeting to a selected tissue. It was shown that the processed Nanopieces were efficiently delivered into cartilage matrix from various species, as well as inside chondrocytes. The delivered Nanopieces were fully functional. A composite of polyethylene glycol (PEG) was used to increase Nanopiece delivery efficiency in a protein-rich environment (such as serum). Rat and mouse models showed that the processed Nanopieces successfully achieved trans-matrix and/or tissue delivery in vivo.
For diagnostics, MMP-13 molecular beacons for disease gene detection were co-delivered with non-targeting scrambled molecular beacons as a non-specific signal negative control and GAPDH molecular beacons as an internal house-keeping gene control. Fluorescence signal was accurately translated into gene expression level exemplary of a non-invasive approach to detect real-time, in-situ gene expression in living animals.
For therapeutics, cytokine (IL-1β) was used to stimulate cartilage degeneration mimicking arthritis, especially rheumatoid arthritis. With Nanopiece delivery of IL-1 receptor siRNA, IL-1 receptor expression was knocked down in chondrocytes in mouse cartilage in vivo, so that cartilage degeneration genes (such as MMP-13, MMP-9) were down-regulated and cartilage anabolic genes (such as Col II) were up-regulated.
Nanopieces were used to deliver ADAMTS-5 siRNA into knee joints of mice with cytokine (IL-1α and retinoic acid) stimulation. Cartilage degeneration was significantly inhibited. To mimic osteoarthritis progression, destabilization of medial meniscus (DMM) was conducted on knee joints of mice. With Nanopiece delivery of ADAMTS-5 siRNA, osteoarthritis progression was prevented. These data indicate the Nanopieces are useful to prevent and/or inhibit cartilage degeneration and arthritis progression.
Example 2
Successful assembly of RNTs into Nanopieces was shown, (see ARROWS) and they were used to deliver various types of cargo reagents including small nucleic acids (siRNA, FIG. 1), long nucleic acids (plasmid DNA, FIG. 2), peptide or protein (Matrilin-3, FIG. 3) as well as small molecules.
Example 2.1
Nanopieces containing SiRNA as cargo were manufactured as follows. 2 μL of a 50 μM siRNA solution was mixed with 10 μL of a 1 mg/mL RNTs mixture. The resulting mixture was sonicated for 60 s. Dilution factors can range from 1 to 50 μL for preparing the siRNA-RNTs complex mixture and sonication times of the resulting mixture can vary from 1 to 600 s. Results are shown in FIG. 1.
Example 2.2
Nanopieces containing DNA were manufactured as follows. 0.5 μg DNA was mixed with 10 μL of a 1 mg/mL RNTs solution. The resulting mixture was sonicated for 60 s. Dilution factors can range from 1 to 50 μL for preparing the DNA-RNTs complex mixture and sonication times of the resulting mixture can vary from 1 to 600 s. Results are shown in FIG. 2.
Example 2.3
Nanopieces containing Matrilin as cargo were manufactured as follows. 10 μL of a 100 μg/mL Matrilin (MATN) protein solution was mixed with 10 μL of a 1 mg/mL RNTs. The resulting mixture was then sonicated for 60 s. Dilution factors can range from 1 to 50 μL for preparing the MAIN-RNTs complex mixture and sonication times of the resulting mixture can vary from 1 to 600 s. Results are shown in FIG. 3.
Example 3
Design and Processing of Nanopieces
FIG. 4 shows an exemplary assembly mechanism. Processing methods were designed before, during and after assembly to manipulate the sizes of Nanopieces. Taking quench and sonication as examples of processing methods before assembly, FIGS. 6 and 7 demonstrate the formation of smaller Nanopieces compared with those generated under standard conditions (FIG. 5). FIGS. 8 and 9 represent size distributions of examples of processing methods during and after assembly. Small Nanopieces were delivered into cells as shown in FIG. 10.
Example 3.1
FIGS. 5A-9B demonstrate Nanopieces of different sizes and width that were imaged under a transmission electron microscope, and their length and width were analyzed with Image J software.
Nanopieces of different lengths and widths were prepared using the following exemplary procedures.
Example 3.1A
5 ug of RNT in 5 uL water was mixed with 50 pmol siRNA in 10 uL water, and then the mixture was sonicated for 2 min to produce Nanopieces (FIGS. 5A and 5B)
Example 3.1B
5 ug of RNT in 5 uL water is heated to 95° C. for 10 min, and then the solution is immediately putted on ice. After totally cooling down to 0° C., RNT solution is mixed with 50 pmol siRNA in 10 uL water, and then the mixture is sonicated for 2 min to produce Nanopieces (FIGS. 6A and 6B).
Example 3.1C
5 ug of RNT in 5 uL water is heated to 95° C. for 10 min, and then the solution is immediately subjected to sonication for 5 min. The resulting RNT solution is mixed with 50 pmol siRNA in 10 uL water, and then the mixture is sonicated for 2 min to produce Nanopieces (FIGS. 7A and 7B).
Example 3.1D
5 ug of RNT in 5 uL in water is mixed with 50 pmol siRNA in 10 uL 0.9% saline, and then the mixture is sonicated for 2 min to produce Nanopieces (FIGS. 8A and 8B).
Example 3.1E
5 ug of RNT in 5 uL in water is mixed with 50 pmol siRNA in 10 uL water, and then the mixture is sonicated for 4 min to produce Nanopieces (FIGS. 9A and 9B).
Example 3.2
FIG. 10 shows that fluorescence labeled RNA was delivered into cells using unprocessed and processed Nanopieces. The Nanopieces were added to chondrocytes and the cells were maintained under standard cell culture conditions for 24 h. Left Panel of FIG. 10 shows unprocessed nanopieces, while the right panel of FIG. 10 shows processed Nanopieces being delivered into cells.
Example 3.3
Various types of Nanopieces and their processing methods are described. Nanotubes are converted into nanorods. As shown in FIG. 4, the use of physical methods (sonication, blending, microwave and/or quenching) or chemical methods (altering pH, adding organic solvents, and/or adding of aromatic chemicals) convert nanotubes into homogenous shorter/longer nanorods to result in shorter/longer Nanopieces compared to standard conditions. (FIGS. 5-7). Nanorods were produced via either sonicating RNTs, or heating RNTs to 90° C., and then quenching them on ice. RNTs or Nanorods were used to form Nanopieces. Nanopieces were characterized using transmission electron microscope and their length and width were analyzed with Image J software.
Example 3.4
Various types of Nanopieces and their processing methods are used to customize the physical characteristics, e.g., length and width, and/or chemical characteristics e.g., surface charge of the delivery vehicle. Two major conditions can be altered: i) assembly conditions (ionic strength, pH and concentration) to achieve Nanopieces with various sizes; and ii) the ratio between nanotubes/nanorods and delivery cargos to achieve different surface charge for the delivery of cargo into different tissues. For example, an increase in ionic strength can be used in the assembly solution to generate longer and wider Nanopieces compared to when using standard conditions (FIG. 4 and FIG. 7). An increase in the ratio of RNTs over siRNA resulted in an increase of the surface positive charge of Nanopieces (FIG. 11). FIG. 8 shows that RNTs and siRNA were dissolved in saline to form Nanopieces as described in the previous sections. Nanopieces were imaged under a transmission electron microscope, and their length and width were analyzed with Image J software. FIG. 11 shows the different ratios of RNTs and siRNA that were used to form Nanopieces. The surface charge (as measured by Zeta potential; mV) of Nanopieces was determined via Nanosizer.
Example 3.5
Processing after assembly included physical methods, e.g., using different power of sonication, heating, blending and/or microwave; or chemical methods, like altering of pH and adding of aromatic chemicals. For example, the use of low, medium and high power of sonication resulted in Nanopieces with different size (length) and morphology (aspect ratio, which is equal to length/width) (FIGS. 4, 56, and 57). FIGS. 56-57 shows that Nanopieces were formed under standard conditions or were processed with different sonication powers (low power is 10% of maximum amplitude of a 700 W sonicator; medium is 50% and high is 100%). Nanopieces were imaged under a transmission electron microscope, and their length and width were analyzed with Image J software.
Example 3.6
Nanopieces are optionally coated. Coating of Nanopieces with PEG facilitated Nanopieces delivery into tissue matrix, especially in a protein-rich environment, such as in the presence of serum (FIG. 20). Although Nanopieces doubled the half-life of delivery cargos (such as molecular beacon, MB) in serum, a covalent linked PEG coating had a 6-time longer half-life than MB only (FIG. 58). Moreover, non-covalent linked PEG only had marginal difference on Nanopieces in terms of stability in serum (FIG. 59). FIGS. 58-59 shows that molecular beacons delivered with/without Nanopieces were soaked in serum. For PEG coating, PEG (MW 400) was either covalently linked or non-covalently coated on Nanopieces. A fluorescence plate read was determined half-life of MBs.
Example 3.6
Nanopieces of different sizes and length were prepared using the following procedure:

    
    
        Step A: Quench before assembly: heating 5 ug RNT in water to 50-99° C. for 10 s-10 mins, then immediately putting it on ice, and mixing with 50 pmol siRNA, then, sonicating for 30 s-2 mins to produce Nanopieces.
        Step B: Sonication before assembly: sonicating 5 ug RNT in water to 50-99° C. for 10 s-10 mins, and mixing with 50 pmol siRNA, then, sonicating for 30 s-2 mins to produce Nanopieces.
        Step C: Increase ionic strength: mixing 5 ug RNT with 50 pmol siRNA in saline, then, sonicating for 30 s-2 mins to produce Nanopieces.
        Step D: Increase sonication time after assembly: mixing 5 ug RNT with 50 pmol siRNA, then, sonicating for 2 mins-10 mins to produce Nanopieces.

Modification of Parameters:

    
    















Size of Nanopieces









Factors
High/Long
Low/Short





Heating
Small
Large


temperature for
(Avg. length
(Avg. length


quench
10 nm~149 nm; Avg. width
150 nm~500 nm; Avg.



diameter 10~29 nm)
width diameter 10~29 nm)


Heating time
Small
Large


for quench
(Avg. length
(Avg. length



10 nm~149 nm; Avg. width
150 nm~500 nm; Avg.



diameter 10~29 nm)
width diameter 10~29 nm)


Sonication time
Small
Large


before
(Avg. length
(Avg. length


assembly
10 nm~149 nm; Avg. width
150 nm~500 nm; Avg.



diameter 10~29 nm)
width diameter 10~29 nm)


Sonication
Small
Large


power before
(Avg. length
(Avg. length


assembly
10 nm~149 nm; Avg. width
150 nm~500 nm; Avg.



diameter 10~29 nm)
width diameter 10~29 nm)


Sonication time
Small
Large


after
(Avg. length
(Avg. length


assembly
10 nm~149 nm; Avg. width
150 nm~500 nm; Avg.



diameter 10~29 nm)
width diameter 10~29 nm)


Sonication
Small
Large


power after
(Avg. length
(Avg. length


assembly
10 nm~149 nm; Avg. width
150 nm~500 nm; Avg.



diameter 10~29 nm)
width diameter 10~29 nm)


Ionic strength
Vary Large
Small



(Avg. length
(Avg. length



150 nm~999micon; Avg.
10 nm~149 nm; Avg. width



width diameter 30~100 nm)
diameter 10~29 nm)























Charge of Nanopieces













Strong/High
Weak/Low







RNT/RNA ratio
Positive
Negative



Negative charge from the
Negative
Positive



cargo (such as RNA other



nucleic acids or proteins)






















Nanopiece
Size
Surface Charge











properties
Small
Large
Negative
Positive





Suitable cells
High and
Low and
Positively
Negatively


or tissues
dense
loose
charged or
charged or



extracellular
extracellular
neutral cell
neutral cell



matrix content
matrix
membrane/
membrane/




content
extracellular
extracellular





matrix
matrix









Example 4
Surface Charge and Matrix/Tissue Binding
Surface charge of Nanopieces were tuned or customized via controlling RNT/delivery cargo ratio (e.g., RNT/siRNA as an example, FIG. 11). Adjusting 4.4 μg ̃30 μg RNTs per 0.1 nmol RNA yielded positively charged Nanopieces. These Nanopieces exhibited excellent binding to negatively charged tissue and/or matrix, as shown in FIG. 12; light grey area and spots are the fluorescence signals from siRNA alone or siRNA. Nanopieces with more than 30 ug RNT per 0.1 nmol RNA are also positively charged. Generally, the ratio will not exceed 30 ug per 0.1 nmol RNA.
Example 4.1
Fluorescence labeled RNA with and without Nanopieces was added onto porcine articular cartilage for 1 h. Then, the cartilage was soaked in HBSS buffer at 37° C. The remaining RNA was analyzed using a fluorescence microscope.
Example 5
Trans-Matrix/Tissue Delivery
Results showed that processed fluorescence labeled siRNA/RNT Nanopieces successfully penetrated into cartilage (FIG. 13). Moreover, it was further demonstrated that GAPDH molecular beacon/RNT Nanopieces not only penetrate into the tissue matrix but also inside cells (FIGS. 14-16). Effective trans-matrix and/or tissue delivery was demonstrated with a variety of species. Light gray areas within FIG. 14-16 around the cell nucleus are the fluorescence signals from molecular beacons.)
Example 5.1
Fluorescence labeled RNA was delivered with and without Nanopieces and was soaked with porcine cartilage. After 24 hours, the cartilage was sectioned and the individual sections were observed under a fluorescence microscope (FIG. 13).
Example 5.2
Fluorescence GAPDH molecular beacon was delivered with and without Nanopieces and soaked with mouse cartilage. After 24 hours, the cartilage was then sectioned and the individual sections were observed under a fluorescence microscope (FIG. 14).
Example 5.3
Fluorescence GAPDH molecular beacon was delivered with and without Nanopieces and soaked with human cartilage. After 24 hours, the cartilage was then sectioned and the individual sections were observed under a fluorescence microscope (FIG. 15).
Example 5.4
Fluorescence GAPDH molecular beacon was delivered with and without Nanopieces and soaked with chicken cartilage. After 24 hours, the cartilage was then sectioned and the individual sections were observed under a fluorescence microscope (FIG. 16).
Example 5.5
Applications of various types of Nanopieces: Various types of Nanopieces can be used for delivery into different tissues or organs as desired. For example, co-injection of small Nanopieces (Avg. length  ̃110 nm, Avg. width  ̃20 nm) (SMALL means Avg. length 10 nm ̃149 nm; Avg. width diameter 10 ̃29 nm) to deliver GAPDH MBs with fluorescence and very large Nanopieces (Avg. length  ̃250 nm, Avg. width  ̃33 nm) (LARGE means Avg. length 150 nm ̃999 micron; Avg. width diameter 30 ̃100 nm) to deliver GAPDH MBs also with fluorescence into knee joints of mice were carried out. Small Nanopieces could be delivered into both cartilage and synovium, while large Nanopieces could only be delivered into synovium (FIGS. 60-61). (Bright area/spots around cell nuclei in FIG. 60-61 are the fluorescence signal from molecular beacons delivered via different sizes of Nanopieces.) Therefore, selective delivery into synovium with processed large Nanopieces was achieved.
Another example was the use of small Nanopieces. Systemic injection of small Nanopieces into mice was carried out. Compared with conventional lipid delivery vehicles, small Nanopieces were found to be able to increase penetration into tissues and organs with dense matrix, which are difficult to infiltrate (such as brain, rib, spine and limb), as well as decreased liver capture (FIGS. 62-63). FIGS. 60-61 shows fluorescence labeled GAPDH molecular beacon delivered with small Nanopieces and also fluorescence labeled GAPDH molecular beacon delivered with large Nanopieces were co-injected into mouse knee joints, and the fluorescence signal was observed under a fluorescence microscope. FIGS. 62-64 shows Far fluorescence labeled GAPDH molecular beacon delivered with Nanopieces or with lipid particles were injected into mice via resto-orbital injection. After 24 hours, the mice were sacrificed and dissected. The fluorescence signal in each organs or tissue was recorded and via a fluorescence molecular tomography.
Example 6
Function
Results showed delivery of Matrilin-3 (MATN3) siRNA/RNT Nanopieces into the mouse cartilage tissue matrix and cells with excellent biological function (FIGS. 17 and 18). Moreover, miRNA-365/RNT Nanopieces were functional, when delivered into human cartilage tissue matrix and cells (FIG. 19). The smaller processed Nanopieces resulted in higher Nanopiece delivery efficacy.
Example 6.1
MATN-3 siRNA was delivered with and without Nanopieces or Lipofectamine 2000 and soaked with mouse cartilage. The MATN-3 gene expression was determined via real time RT-PCR (FIG. 17).
Example 6.2
MATN-3 siRNA was delivered with unprocessed or processed Nanopieces and was soaked with mouse cartilage. The MATN-3 gene expression was determined via real time RT-PCR (FIG. 18).
Example 6.3
Various doses of miR-365 (0.1, 0.5 and 1.0 nmol) were delivered with Nanopieces and were soaked with human cartilage. The miR-365 expression was determined via real time RT-PCR (FIG. 19).
Example 7
Compositions
FIG. 20 shows that a composite of PEG increases Nanopiece delivery efficiency in a protein-rich environment (such as serum).
Example 8
In Vivo Delivery
FIGS. 21 and 27 show injection of Nanopieces into an articulating joint. Injection of GAPDH molecular beacon/RNT Nanopieces into knee joints of a mouse (FIG. 21) resulted in a significant fluorescence signal compared with beacon only (in the absence of RNT Nanopieces). The signal lasted more than 2 weeks in the knees (FIGS. 22-24). In rats, a significant fluorescence signal was also obtained by injecting GAPDH molecular beacon/RNT Nanopieces into knee joints. The fluorescence signal was robust after washing out the adhered fluorescence molecules on the articular surface (FIGS. 25-26). Matrilin-3 siRNA Nanopieces were injected into knees of baby one-week-old mice and was found to be functional. Histology slides of cartilage sections confirmed the successful delivery of the Nanopieces (FIG. 28; light grey areas around the cell nuclei illustrate the fluorescence signal from molecular beacons. Effective in vivo trans-matrix/tissue delivery of processed Nanopieces (Nanopieces) was demonstrated in these experiments.
Example 8.1
Fluorescence labeled GAPDH molecular beacon was delivered with and without Nanopieces and injected into mouse knee joints. The fluorescence signal was recorded via fluorescence molecular tomography (FIGS. 22-24).
Example 8.2
Fluorescence labeled GAPDH molecular beacon was delivered with and without Nanopieces and injected into rat knee joints. The fluorescence signal was recorded via fluorescence molecular tomography (FIGS. 25-26).
Example 8.3
Fluorescence labeled GAPDH molecular beacon was delivered with and without Nanopieces and injected into baby mouse knee joints. The mouse was sacrificed and knee joint was sectioned for observation under a fluorescence microscope (FIGS. 27-28; light grey areas around the nuclei in FIG. 28 illustrate the fluorescence signal from molecular beacons.
Example 9
Diagnostics
To detect OA progression, MMP-13 was selected as a target gene. MMP-13 molecular beacon was designed and its function validated in vitro. As shown in FIG. 29, MMP-13 molecular beacon was delivered by methods described herein and found to emit fluorescence in chondrocytes after stimulation. Light areas shown in FIG. 29 illustrate the fluorescence signal from molecular beacons. The MMP-13 molecular beacon was prepared according to the following procedure:

    
    
        Step one: Pre-heat RNT nanotubes solution, then quench it by placing tube on ice.
        Step two: Sonicate RNT nanotubes solution.
        Step three: Dilute MMP-13 molecular beacon or IL-1beta receptor siRNA in water, then mix with RNT nanotubes solution in a certain ratio (50 pmol siRNA or 100 pmol molecular beacon to 5 ug RNT), then vertex well.
        Step four: Sonicate the mixture described in Step three, then spin all liquid down. MMP-13 molecular beacon or IL-1beta receptor Nanopieces was assembled after Step four.

*Standard preparation only includes Step three and Step four. Joint preparation includes all steps.

    
    


For in vivo diagnosis, the medial meniscus (DMM) was destabilized to induce OA on one knee of the mice, whereas on the other knee a sham surgery was performed. Right after surgery, MMP-13 molecular beacon was delivered for target gene detection together with a non-targeting scrambled molecular beacon as a non-specific signal serving as a negative control. In addition a GAPDH molecular beacon for an internal house-keeping gene control was also administered. After 4 days, the knee with OA induction, showed a significantly stronger signal than the sham knee (FIG. 30). Moreover, using such a real-time, in-situ, non-invasive diagnosis approach, the signals between DMM and sham were quantitatively compared in a time-depend curve (FIG. 31). Methods were provided to continuously monitor a specific gene expression during OA progression in living animals. Moreover, animals were sacrificed at day 4 and day 11 to determine their MMP-13 expression level via real time RT-PCR. Results showed that the non-invasive diagnostic technology described herein accurately detected gene expression level compared with PCR (FIG. 32).
Fluorescence and histology analysis showed that the damaged articular cartilage surface was the area emitting fluorescence signal from MMP-13 molecular beacon (FIGS. 37-38). In FIG. 37, ARROWs indicate the fluorescence signal as a result from MMP-13 molecular beacon. In FIG. 38, the dark grey color in articular cartilage was aggrecan staining. DMM surgery resulted in loss of aggrecan staining and damage to articular cartilage.
In addition to MMP-13, ADAMTS-5 molecular beacon for OA diagnosis was also shown. Again, the ability of this molecular beacon to detect ADAMTS-5 gene expression in vitro was demonstrated (FIGS. 39-41; light grey areas around the cell nuclei in FIG. 39-41 are the fluorescence signal from molecular beacons. RED channel showed signal from GAPDH beacons; while GREEN channel showed signal from ADAMTS-5 or Scrambled beacons. The up-regulation pattern of ADAMTS-5 during OA development was also shown (FIGS. 42-43).
These data indicate that the methods are useful for accurate and specific gene expression detection, thereby permitting reliable diagnosis in a real-time, in-situ and in a non-invasive manner in living animals.
Example 9.1
Fluorescence labeled GAPDH molecular beacon and fluorescence labeled MMP-13 molecular beacon or fluorescence labeled scrambled molecular beacon delivered with Nanopieces was added into chondrocytes under standard cell culture conditions or stimulated with 10 ng/mL IL-1β (FIG. 29).
Using an established method (Tyagi et al Nat. Biotech, 1998, 16:49-53), MBs were designed to target mouse MMP-13 or GAPDH mRNA with a fluorophore/quench pair. Scramble sequence MB (Scramble) was verified to not bind with any mouse mRNA via BLAST. In vitro delivery and validation: MBs were delivered into chondrocytes by Nanopieces. Specifically, after stimulation with IL-1β for 24 hours, chondrocytes were co-transfected GAPDH and scramble MBs or GAPDH and MMP-13 MBs via Nanopieces. Real time RT-PCR and fluorescence microscopy were used to verify the stimulation of MMP-13 expression and the successful fluorescence signal resulted from MMP-13 MB.
To test the efficacy of mRNA detection in chondrocytes using MBs delivered by Nanopieces, primary mouse chondrocytes were transfected with MBs either with or without IL-1β treatment. Before IL-1β treatment, the housekeeping GAPDH MB was detected while the MMP-13 MB was not (FIG. 29, left panels). In contrast, after IL-1β treatment, both GAPDH MB and MMP-13 MB were detected, indicating the induction of MMP-13 mRNA levels by IL-1β (FIG. 29, right panels). Realtime rtPCR showed that MMP-13 mRNA level was up-regulated by about 10 times upon IL-1β stimulation. In contrast, Scramble MB transfection did not show any fluorescence, indicating that the fluorescence of MMP-13 MB was not due to non-specific degradation.
Example 9.2
Fluorescence labeled GAPDH, MMP-13 and Scrambled molecular beacon delivered with Nanopieces was injected into mouse knee joints after destabilization of medial meniscus (DMM) surgery or Sham surgery, and then the fluorescence signal was recorded and analyzed via a fluorescence molecular tomography (FIGS. 30-31). DMM or sham surgeries were performed on 10-week-old 129SVE male mice to induce osteoarthritis. One week after surgery, MMP-13 and scramble MBs with different fluorophores delivered by Nanopieces were injected into knee joints of mice. Small animal fluorescence molecular tomography (FMT) was used to determine the fluorescence signal that resulted from MMP-13 expression in the live animals for 3 weeks. The Scramble MB showed low fluorescence in both DMM and Sham surgery knee joints. After subtracting Scramble MB basal level signals, MMP-13 MB real signal was about 40 times stronger in the DMM leg than the sham leg (FIGS. 50, and 54-55). Such MMP-13 MB signals persisted, even for 3 weeks after injection of MBs.
Example 9.3
Mouse knee joint cartilage was isolated 4 days or 10 days after DMM or Sham surgery, and MMP-13 expression was determined via real time RT-PCR (FIG. 32).
Example 9.4
Fluorescence labeled MMP-13 molecular beacon and Scrambled molecular beacon delivered with Nanopieces was injected into mouse knee joints after DMM or Sham surgery. After 30 days, the animals were sacrificed and their knee joints were sectioned for histology and fluorescence scan (FIGS. 37-38).
Example 9.5
Fluorescence labeled GAPDH molecular beacon, fluorescence labeled ADAMTS-5 molecular beacon or fluorescence labeled Scrambled molecular beacon delivered with Nanopieces was added into chondrocytes under standard cell culture conditions or stimulated with 10 ng/mL IL-1α and 10 μM retinoic acid (FIGS. 39-41).
Example 9.6
Fluorescence labeled GAPDH, ADAMTS-5 and Scrambled molecular beacon delivered with Nanopieces was injected into mouse knee joints after DMM or Sham surgery, and then the fluorescence signal was recorded and analyzed via a fluorescence molecular tomography (FIGS. 42-43). FIG. 42 shows a stronger fluorescence signal resulting from ADAMTS-5 molecular beacon in DMM surgery leg than Sham leg. FIG. 43 shows the pattern of ADAMTS-5 expression after surgery.
Example 10
Therapeutics
IL-1 receptor (IL-1R) siRNA/Nanopieces were injected into one knee of mice and non-targeting scrambled siRNA/Nanopiece was injected into the other knee. Cartilage degeneration was stimulated with catabolic cytokine (such as IL-1β) in both knees mimicking an inflammation environment during arthritis. Successful knock down of IL-1R in chondrocytes in mouse cartilage was observed with Nanopiece delivery of IL-1R siRNA in vivo (FIG. 33). Moreover, cartilage degeneration genes (such as MMP-13 and MMP-9, FIG. 33) were down-regulated and cartilage anabolic genes (such as Col II, FIG. 33) were up-regulated.
Nanopieces were used to deliver ADAMTS-5 siRNA into knee joints of mice that had been treated with cytokines (IL-1α and retinoic acid). Results showed that cartilage degeneration and aggrecan cleavage was significantly inhibited after ADAMTS-5 siRNA treatment (FIG. 34). In the top two panels, the dark grey color in articular cartilage was aggrecan staining. Without ADAMTS-5 siRNA treatment, aggrecan staining is weaker than the treatment group, indicating loss of aggrecan. In the bottom two panels, dark staining around the cell nuclei was epitope staining from aggrecan cleavage. Without ADAMTS-5 siRNA treatment, the staining is stronger than the treatment group, indicating cleavage of aggrecan.
To mimic osteoarthritis progression, DMM surgery on knee joints of mice was conducted. Osteoarthritis progression was shown to be prevented or slowed with Nanopiece delivery of ADAMTS-5 siRNA (FIGS. 35 and 36). In FIG. 35, the dark grey color in articular cartilage was aggrecan staining. A RROWs point out loss of aggrecan staining or damage to articular cartilage in the groups without ADAMTS-5 siRNA treatment; while with treatment, there was very little loss of aggrecan or damage to articular cartilage. Also, immunohisology results showed that aggrecan cleavage was inhibited with delivery of ADAMTS-5 siRNA (FIG. 46). In FIG. 46, dark staining around cell nuclei was epitope staining from aggrecan cleavage. Without ADAMTS-5 siRNA treatment, the staining is stronger than the treatment group, indicating cleavage of aggrecan.
In addition, ADAMTS-5 siRNA was delivered via Nanopieces to human cartilage ex vivo. Protection of human cartilage from cytokine-induced cartilage degradation was demonstrated (FIGS. 44-45). In FIG. 44, dark staining around cell nuclei was epitope staining from aggrecan cleavage. Without ADAMTS-4 or 5 siRNA treatment, the staining is stronger than the treatment group, indicating cleavage of aggrecan. In FIG. 45, dark color in articular cartilage was aggrecan staining. Without ADAMTS-4 or 5 siRNA treatment, aggrecan staining is weaker than the treatment group, indicating loss of aggrecan.
These data indicate that the methods are useful to prevent and/or inhibit cartilage degeneration and arthritis progression.
Example 11
Synthesis
Example 11.1
RNTs and TBLs to form Nanopieces are made by first synthesizing a module [(e.g., compound of Formula I or compound of Formula II, respectively]. Nanotubes (RNTs or TBLs) are then processed (Processing-1, Processing-2) to make nanorods and Nanopieces, respectively (see, e.g., FIG. 53). A module for making a Nanopiece was synthesized according to methods described in U.S. Pat. No. 6,696,565 and subsequently purified prior to using the same in the preparation of functional Nanopieces. Liquid chromatography purification was used to purify the synthetic products derived from Formula I and/or Formula II to ensure the success of forming functional and low toxic Nanopieces. In liquid chromatography, trifluoroacetic acid (TFA) is usually applied to keep an acidic eluent environment. Due to known toxicity of TFA or fluoride residual, which made isolated materials undesirable for preclinical and clinical studies, a modification to include hydrochloric acid (HCl) or phosphoric acid during the purification process was developed as an alternative TFA.
Liquid chromatography was performed on C18 reverse-phase column, and agilent 1260 Infinity Quaternary HPLC System was used. One example of gradient used in isolation is shown below:













Time











0 min
10 min
15 min
















Percentage of Solvent A
90
65
0



Percentage of Solvent B
0
25
90



Percentage of Solvent C
10
10
10










*Solvent A is H2O, Solvent B is 100% acetonitrile, and Solvent C is 0.05N hydrochloric acid. The cell toxicity was evaluated using a standard cell viability test. ATDC5 cells were treated with RNTs, and after 48 hours cell viability normalized to negative controls (as 100). Results are showed in FIG. 47. These results demonstrate successful isolation of modules using a modified HPLC purification method to obtain RNTs. Using HCl instead of TFA in this purification process avoided the presence of fluorine containing contaminates within the module, which contributed to the toxicity of the resulting nanotube. Thus, use of HPLC decreased the toxicity of RNTs and use of HCl versus TFA further decreased the cytotoxicity. Molecular modules, e.g, TBLs were therefore isolated by applying HCl in liquid chromatography purification. This purification scheme is applicable for module I compounds (for RNT assembly and for module 11 compounds for TBL assembly) to yield functional Nanopieces with low toxicity.

Example 11.2
Conversion of nanotubes (such as RNTs and TBLs) into nanorods was accomplished according to a process called “processing-1” (FIG. 53). In Processing-1, nanotubes are converted into short and homogeneous nanorods. This is very important to produce Nanopieces small enough to penetrate some types of tissue matrices for introduction of therapeutics into the tissue. Conversion of nanotubes to nanorods can be accomplished by altering pH, temperature, and/or using physical methods (such as sonicating, heating and blending (e.g. homogenizer)), and/or addition of aromatic chemicals. Different sizes of Nanopieces can be produced (FIGS. 5, 6 and 48). Based on the Nanopiece assembly mechanism, the processing approach may include at least one of the following: 1) before assembly, controlling the length and bundle of RNTs via changing physical and/or chemical conditions such as temperature, molecule motion and/or vibration (like sonication) and pH; 2) during assembly, adjusting assembly conditions via changing physical and/or chemical conditions including concentrations, pH and ionic strength to enhance and/or reduce the formation and stacking of Nanopieces; 3) after assembly, breaking long or stacked Nanopieces by via changing physical and/or chemical conditions including enhancing molecule motion/vibration (like sonication).
Example 11.3
Preparation of Nanopieces was accomplished by a process called “processing-2” (FIG. 55). Processing-2 occurs after the incorporation between nanotubes or nanorods with delivery cargo and formation of bundles, ribbons or other agglomerates. These agglomerates can then be transformed to Nanopieces (FIG. 49). The size of the Nanopieces can be changed with changes in pH, ionic strength, temperature and concentration (FIGS. 4, 7-9).
FIGS. 15-23 and 26-32 demonstrated the successful tissue delivery after combining the above methods in Examples 11.1-11.3.
Example 11.4
Preparation of small and large lipid Nanoparticles was accomplished using the procedures described below.
Preparation of large lipid nanoparticles with IL-1R siRNA (sphere shape 110 nm to 180 nm diameter):

    
    
        1) Dissolve siRNA in 20 mM citrate buffer (pH 5.0, nuclease free) to achieve a concentration of 50 μM.
        2) Dissolve DSPC, cholesterol, DODMA, and DSG-PEG (20:48:2:30 molar ratio) in absolute, anhydrous ethanol, and then add nuclease free water to achieve a concentration of 90% ethanol.
        3) The total concentration of lipid in solution is then adjusted to 20 mM.
        4) 1 μL of siRNA and 1 μL of lipid solutions are heated to 37° C., then mix at the same temperature and dilute with 8 uL nuclease free water. Sit at least 30 minutes before use.

Preparation of Small Lipid Nanoparticles with IL-1R siRNA (Sphere Shape 70 nm to 120 nm Diameter):

        1) Dissolve siRNA in 10 mM citrate, 30 mM NaCl (pH 6.0, nuclease free) to achieve a concentration of 50 μM.
    
    


2) Dissolve DSPC, DSG-PEG, cholesterol, SPDiOC18, and DOTMA (10:10:39.8:0.2:40 molar ratio) in absolute, anhydrous ethanol, and then add an aqueous buffer (50 mM citrate, pH 4.0, nuclease free) to achieve a final concentration of 40% ethanol.

    
    
        3) The total concentration of lipid in solution is then adjusted to 20 mM.
        4) Extrude the lipid solution through two nucleopore polycarbonate filters (100 nm, 10 passes).
        5) 1 μL extruded lipid solution and 1 μL siRNA are mixed under constant vortex, then dialyzed in PBS overnight to increase the pH to about 7.4.

FIG. 67 shows successful localization/delivery of cargo to cartilage tissue using nucleic acid-loaded lipid nanoparticles. The small siRNA lipid nanoparticles localized to, penetrated cartilage tissue, and inhibited expression of the target gene.

    
    


Example 11.5
Preparation of small and large polymer Nanoparticles was accomplished using the procedures described below.
Preparation of Large and Small Polymer Nanoparticles with IL-1R siRNA:
1) Dissolve poly-lysine (PLL) (molecular weight, 15 kDa-30 kDa) in nuclease free water to 0.2 mg/mL.
2) Dialyze to remove salt (HBr).
3) Lyophilize.
To Prepare Large PLL/siRNA Nanoparticles (100-250 nm Diameter):
1) Dissolve siRNA and PLL in 0.15M NaCl to concentrations of 10 μM and 25 μM, respectively.
2) Quickly add 1 uL 50 μM siRNA solution to 15 uL 100 μg/mL PLL and pipette well at room temperature.
3) Pipette and let sit for at least 30 minutes before use.
To Prepare Small PLL/siRNA Nanoparticles (50-75 nm Diameter):
1) Dissolve siRNA and PLL in nuclease free water to concentrations of 50 μM and 100 μg/mL, respectively.
2) Quickly add 1 uL 50 μM siRNA solution to 15 uL 100 μg/mL PLL and pipette well at room temperature.
3) Use within 30 minutes of reaction.
FIG. 68 shows successful localization/delivery of cargo to cartilage tissue using nucleic acid-loaded polymer nanoparticles. The small siRNA polymer nanoparticles localized to, penetrated cartilage tissue, and inhibited expression of the target gene.
FIGS. 67 and 68 demonstrated the successful tissue delivery of the above prepared lipid or polymer nanoparticles. Animals were injected with prepared large/small lipid or polymer nanoparticles delivered with IL-1R siRNA to right knees of mice. (Animal left knees were used as negative controls). After 24 hours, euthanize animals were euthanized and their knee cartilage was collected for real time RT-PCR. These data indicate that cargo-loaded nanostructures such as RNTs comprising compounds of Formula I, TBLs comprising compounds of Formula II, as well as lipid nanoparticles, and polymer nanoparticles successfully deliver cargo to target tissues.
Example 12
A Non-Invasive, Early, and Sensitive Detection of Osteoarthritis Through In Vivo Imaging of MMP-13 mRNA Levels by Molecular Beacon (MB) and Nanopiece Delivery Technology
MBs were designed to target MMP-13 or GAPDH mRNA with a fluorophore/quench pair using a mouse model. Scramble sequence MB (Scramble) was verified to not bind with any mouse mRNA via BLAST. To demonstrate in vitro delivery and validation; MBs were delivered into chondrocytes by Nanopieces. After stimulation with IL-1β for 24 hours, chondrocytes were co-transfected GAPDH (red) and scramble (green) MBs or GAPDH (red) and MMP-13 (green) MBs via Nanopieces. Real time RT-PCR and fluorescence microscopy were used to verify the stimulation of MMP-13 expression, and a successful fluorescence signal resulted from using a MMP-13 MB.
Destabilization of the medial meniscus (DMM) surgery and in vivo delivery: DMM or sham surgeries were performed on 10-week-old 129SVE male mice to induce osteoarthritis. One week after surgery, MMP-13 and scramble MBs with different fluorophores delivered by Nanopieces were injected into knee joints of mice. Small animal fluorescence molecular tomography (FMT) was used to determine the fluorescence signal resulted from MMP-13 expression in the live animals for 3 weeks.
To test the in vitro efficacy of mRNA detection in chondrocytes using MBs delivered by Nanopieces, primary mouse chondrocytes were transfected with MBs either with or without IL-1β treatment. Before IL-1β treatment, the housekeeping GAPDH MB (red) was detected while the MMP-13 MB (green) was not. In contrast, after IL-1β treatment, both GAPDH MB (red) and MMP-13 MB (green) were detected, indicating the induction of MMP-13 mRNA levels by IL-1β. Realtime rtPCR showed that MMP-13 mRNA level was up-regulated by about 10 times upon IL-1β stimulation. In contrast, Scramble MB transfection did not show any green fluorescence, suggesting that the fluorescence of MMP-13 MB was not due to non-specific degradation.
To evaluate in vivo efficacy, the following studies were carried out. After DMM surgery, MMP-13 MB was delivered intra-articularly to the knee joint of adult mice with Scramble MB that emits fluorescence at a different wave length than MMP-13 MB. Only a week after surgery, the DMM surgery leg displayed a strong MMP-13 signal than the contralateral Sham surgery leg (FIG. 2, left panel). In contrast, the Scramble MB showed very low fluorescence in both DMM and Sham surgery knee joints. After subtracting Scramble MB basal level signals, MMP-13 MB real signal was about 40 times stronger in the DMM leg than the sham leg. Such MMP-13 MB signals persist, even for 3 weeks after injection of MBs.
MMP-13 MB delivered by Nanopiece technology represents a sensitive tool to detect pro-inflammatory degenerative conditions as evidenced with chondrocytes in vitro and in OA animal models in vivo. This technology detects pathogenesis of OA at an early stage (within a week) in a mild OA model (DMM). A high sensitivity was achieved due to the detection at the mRNA level and the high efficiency of MB intracellular delivery by Nanopieces. The combination of molecular beacon and Nanopieces technology provided a powerful tool for early detection of OA in vivo in a specific and sensitive manner without harming any joint tissues.
Matrix metalloproteinases (MMP) are the major enzymes that degrade the components of the extracellular matrix during arthritis progression. MMP-13, which is usually produced by cartilage and bone, degrade interstitial collagens (types I, II and III) in both OA and RA. Expression of MMP-13 is low in normal cells, whereas in pathologic condition excess MMP-13 production is associated with inflammation. Thus, mRNA level of MMP-13 is useful as a diagnostic and prognostic tool for assessment of arthritis development. Therefore MMP-13 is recognized as a reliable target in early diagnosis of arthritis. These data indicate that intra-articular injection of Nanopieces+payload were successfully introduced into joint tissue and that the payload was functionally active after delivery.
The system and compositions described herein overcame the difficulty of accurately translating molecular beacon signal into MMP-13 mRNA expression level. MMP-13 upregulation pattern was demonstrated during OA progression using the Nanopiece—delivered beacons. Compared to earlier and current research and clinical methods, Nanopiece-Molecular Beacon technology achieved much earlier and more sensitive detection.
Given the benefit of the above disclosure and description of exemplary embodiments, it will be apparent to those skilled in the art that numerous alternative and different embodiments are possible in keeping with the general principles of the invention disclosed here. Those skilled in this art will recognize that all such various modifications and alternative embodiments are within the true scope and spirit of the invention. While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description is to be considered as exemplary and not restrictive in character, it being understood that, only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. The appended claims are intended to cover all such modifications and alternative embodiments. It should be understood that the use of a singular indefinite or definite article (e.g., “a,” “an,” “the,” etc.) in this disclosure and in the following claims follows the traditional approach in patents of meaning “at least one” unless in a particular instance it is clear from context that the term is intended in that particular instance to mean specifically one and only one. Likewise, the term “comprising” is open ended, not excluding additional items, features, components, etc. References identified herein are expressly incorporated herein by reference in their entireties unless otherwise indicated.

## Claims

1. A nanopiece for selective drug delivery to a bodily tissue comprising a compound of formula I or formula II or a combination thereof, and a nucleic acid:



wherein, X is CH or N;
R2 is hydrogen or a linker group;
Y is absent when R2 is hydrogen or is an amino acid side chain, amino acid or polypeptide; and
R1 is hydrogen or aliphatic,
wherein the nanopiece has a size in at least one dimension between 0.1 nm and 150 nm,
wherein a ratio of the compound to nucleic acid ranges from 4.4 to 30 μg compound per to 0.1 nmol of the nucleic acid,
wherein the nanopiece is positively charged at pH 7-7.5.

2. The nanopiece of claim 1, wherein the nucleic acid is a diagnostic agent or a therapeutic agent.

3. The nanopiece of claim 1, wherein the nanopiece has a net positive charge of a Zeta potential >+8 mV.

4. The nanopiece of claim 1, wherein the compound is selected from:

5. The nanopiece of claim 1, wherein R2 comprises an amino acid side chain or is selected from:

6. The nanopiece of claim 1, wherein the compound is selected from

7. The nanopiece of claim 1, wherein R1 is C1 to C10 alkyl.

8. The nanopiece of claim 1, wherein the nucleic acid comprises siRNA.

9. The nanopiece of claim 1, wherein the nucleic acid comprises a molecular probe or a molecular beacon.

10. The nanopiece of claim 8, wherein the nucleic acid comprises an IL-1 receptor siRNA.

11. The nanopiece of claim 9, wherein the nucleic acid is the molecular beacon detecting MMP-13 or ADAMTS-5.

12. The nanopiece of claim 1, further comprising one or more analgesic agents, anti-inflammatory agents, immunosuppresive agents, antifungal agents, antibiotic agents, lubricants, anti-cancer agents, NMDA receptor antagonists, or antiviral agents.

13. A method of making a nanopiece of claim 1, comprising:
combining the compounds of Formula I or II and the nucleic acid, wherein the compounds form a nanotube and the nanotube and the nucleic acid form a complex; and
processing the complex comprising the nanotube and the nucleic acid to yield the positively charged nanopiece, said nanopiece comprising a size in at least one dimension of between 0.1 nm and 150 nm.

14. A composition comprising a nanopiece of claim 1 for selective delivery of a therapeutic drug or diagnostic agent to a target bodily tissue.

15. The nanopiece of claim 1, wherein the nanopiece comprises a size of ≦30 nm in at least one dimension.

16. The nanopiece of claim 1, wherein the nanopiece has a size of 1 to 30 nm in at least one dimension.

17. The nanopiece of claim 1, wherein a ratio of the compound to nucleic acid ranges from 4.4 to 30 μg compound per to 0.1 nmol of the nucleic acid.

18. The nanopiece of claim 8, wherein the nucleic acid comprises ADAMTS-5 siRNA.

19. The nanopiece of claim 8, wherein the nucleic acid comprises Matrilin-3 siRNA.

20. The nanopiece of claim 8, wherein the nucleic acid comprises a siRNA targeting a FGF receptor.

21. The nanopiece of claim 1, wherein the nucleic acid comprises microRNA-365.

22. The nanopiece of claim 1, wherein the nucleic acid comprises microRNA-1.46a.

23. The nanopiece of claim 1, wherein the nucleic acid comprises microRNA-1.40.

24. The nanopiece of claim 1, wherein the nucleic acid comprises an anti-microRNA.

25. The method of claim 13, wherein the processing comprises altering pH, ionic strength or temperature, sonicating, heating and blending of the nanotube and the nucleic acid.

26. The method of claim 13, wherein the processing is sonicating.