Therapeutic Antibodies – Richard H. Weisbart, US Department of Veterans Affairs VA
Abstract for “Intranuclear Protein Transduction Through a Nucleoside Salvage Pathway”
“Provided are conjugate molecules that contain a substrate for the nucleoside transportation pathway linked to an activate agent. The conjugate can then be transported into a cell, or into the nucleus via a cellular Nucleoside Transport pathway. Methods of delivering a conjugate to a target cell that expresses a nucleoside transportation pathway are also provided. The conjugate molecules contain a substrate for the active agent’s nucleoside pathway. Methods for screening conjugates transported via nucleoside transport pathways are also provided. A method of treating a patient with a disorder or disease that affects tissues that express nucleoside transport pathways is also provided. In this case, a conjugate containing an effective agent for treating the disorder can be administered to the patient. A method of treating an autoimmune disorder involves administering a compound that blocks a nucleoside transportation pathway to the patient.
Background for “Intranuclear Protein Transduction Through a Nucleoside Salvage Pathway”
“1. “1.
“The invention concerns conjugate molecules, and more specifically to conjugate molecule’s use in the delivery active agents to cells via endogenous cellular transportation pathways.
“2. Background Information”
“Transporter proteins play a role in the cellular uptake and/or passage of different molecules into and through cells. Carrier-mediated transport systems are proteins that are anchored to the cell’s membrane by multiple membrane-spanning domains. They function by transporting substrates through active and passive mechanisms. Carrier-mediated transport systems play an important role in the active and passive, facilitated transportation of important nutrients like vitamins, sugars, as well as amino acids. Organs like the liver and kidney also have carrier-mediated transporters. These proteins are involved with the excretion and re-absorption circulating compounds. The lipid bilayers of cellular membranes are often unable to diffuse hydrophilic or polar compounds. There are specific carriers-mediated transporters that allow active transport of small molecules, such as amino acids, di-, tripeptides, nucleosides, monosaccharides and water-soluble vitamins, across biological membranes.
“Nucleoside transporters are the primary carriers that mediate the uptake and release of physiological nucleosides, as well as many of their synthetic counterparts, by mammalian cells. There are two types of nucleoside transporters: (i), equilibrative (facilitated dispersion) and (ii), concentrative (secondary activate) sodium-dependent. Two equilibrative transport systems with similar broad substrate specificities have been identified and designated as the es (equilibrative sensitive) and ei (equilibrative insensitive) transporters, on the basis of their sensitivity or insensitivity to inhibition by nitrobenzylthioinosine (NBMPR, 1), respectively. Functionally, six sodium ion-coupled nucleoside transporters (concentrative) have been identified in mammalian tissues. They are cif/N1, cib/N3, cib/N3, cs/N5, cs/N6 and cs/N5.
“The anti-DNA antibody fragment3E10 Fv has been shown to be a novel molecular transport vehicle. It penetrates living cells with specific nuclear location, lack of toxicity, and delivers therapeutic cargo proteins in vitro as well as in vivo. To develop new molecular therapies that rely on intranuclear macromolecule transduction, it is crucial to understand the mechanism by which 3E10 Fv crosses cell membranes.
“The invention was based upon the discovery that a DNA binding antibody can penetrate cells and locate in the nucleus. It is transported via a nucleoside transport path.”
“Accordingly to one embodiment, conjugates are provided that include a substrate capable of being transported via a nucleoside transportation pathway and an active agent linked with the substrate. The conjugate is then transported using the nucleosidetransport pathway. Particularly in embodiments where the substrate is an antibody, the antibody is not monoclonal antibody 3E10 (or a fragment thereof). In some embodiments, the nucleoside transportation pathway includes either an equilibrative or concentrative nucleosidetransporter. In embodiments in which the nucleoside transport pathway involves an equilibrative nucleoside transporter, such a transporter may be insensitive to inhibition by low concentrations of nitrobenzylmercaptopurine riboside (NBMBR).”
“Another embodiment of the invention provides methods for delivering a conjugate into a target cell that expresses a nucleoside transportation pathway. These methods involve contacting a target cell that expresses the nucleoside transportation pathway with a conjugate, which includes a substrate capable of being transported via a nucleoside pathway and an active agent linked thereto. The conjugate is then transported through the nucleoside pathway. Particularly in embodiments where the substrate is an antibody, the antibody must not be the monoclonal antibody3E10 or fragment thereof. In some embodiments, the nucleoside transportation pathway includes either an equilibrative or concentrative nucleosidetransporter. In embodiments in which the nucleoside transport pathway involves an equilibrative nucleoside transporter, such a transporter may be insensitive to inhibition by low concentrations of nitrobenzylmercaptopurine riboside (NBMBR).”
“A further embodiment of the invention provides methods for screening conjugates for transport via a nucleoside transportation pathway. These methods involve contacting a cell that expresses the nucleoside transportation pathway with a conjugate in suitable conditions for transport; and determining if the conjugate has been transported into the cell via the nucleoside pathway. In some embodiments, the determining step involves comparing the amount conjugate transported into cells expressing the nucleoside transportation system with the amount transported into control cells that do not express the nucleosidetransport system. These embodiments indicate that transport is via the nucleoside pathway if there is an increase in conjugate transport from the cell expressing it.
“Another embodiment of the invention provides methods for treating a disease in a cell or tissue that expresses a nucleoside transportation pathway. This method involves administering to the patient with the disease or disorder a conjugate that includes a substrate capable of being transported via the nucleoside transportation pathway and an active agent for treatment. The nucleoside pathway also transports the conjugate. The active agent is delivered to the cells and tissues by the conjugate being transported there. The active agent is delivered to the affected cells if the disease or disorder involves the skeletal muscles in certain embodiments.
“According yet another embodiment, methods are available for treating a gene disorder. The method involves administering a conjugate to a patient with a genetic disease. A substrate is capable of being transported via an equilibrative nuclear transporter. Further, the active agent is linked and the conjugate is then transported by the equilibrative nuclear transporter. Cells then receive the active agent. The active agent may be a gene or protein that is missing in patients with the genetic disorder.
“In some other aspects, the disclosure offers a method for treating cancer. The method involves administering a conjugate to a patient with cancer. A substrate is capable of being transported via an equilibrative nuclear transporter. Further, the conjugate can be transported via the equilibrative nuclear transporter. This allows the conjugate to be transported into cancerous cells and delivers the active agent. The active agent may be a tumor suppressor gene, tumor suppressor protein or even a tumor suppressor gene.
“Accordingly to another embodiment of the invention, methods for treating an autoimmune condition include administering to a patient with the disorder a compound that blocks transport via a nucleoside transport route.”
“The present disclosure also includes a pharmaceutical composition that contains a conjugate as described herein, and an agent that promotes ENT2 transcription in a tissue. The agent that promotes ENT2 expression is, in some instances, an agent that treats or inhibits Hypoxia or that inhibits HIF-1. It could be hypoxic tissue such as a hypoxic tumour, a tissue that has insufficient blood supply, an ulcer, diabetic ulcer, poorly healing wounds, an ischemic region, an area that is ischemic after stroke or an area that is ischemic from cardiovascular disease. The agent that inhibits HIF-1 may be used in certain embodiments. A siRNA, an RNaseI construct, or a miRNA which reduces HIF-1? expression. The HIF-1 may be used in some embodiments. HIF-1 is a chemotherapeutic agent, including topotecan (NSC 644221), PX-478, 17-AAG or bevacizumab. In some embodiments, the agent that treats hypoxia or inhibits it is one that normalizes tumor vasculature or alters the redox status of a tissue. Hypoxia can be treated or inhibited by excess oxygen, TSC or almitrine.
“Furthermore is provided a treatment method for ENT-2 deficient tissues. It involves: a) administering an agent to promote ENT2 expression/activity and b) administering one the conjugates herein.
“Before the methods are described, it should be understood that this invention does not limit to specific compositions, methods, or experimental conditions. As such compositions and methods may vary. The terminology used in this document is intended to describe particular embodiments and is not meant to limit the invention’s scope.
“As used herein and in the appended claims, singular forms?a,?an, and??the” If the context requires otherwise, plural references should be included. For example, a reference to “the method” could be used. Includes one or more methods and/or steps described herein that will be apparent to persons skilled in art upon reading this disclosure.
“Unless otherwise stated, all technical terms and scientific terms used in this invention have the same meanings as those commonly understood by an ordinary skilled person of the art to which it belongs.”
“According to the present invention, conjugates are provided that contain a substrate capable of being transported via a nucleoside transportation pathway and an active agent linked with the substrate. The conjugate is then transported using the nucleoside pathway. This allows the conjugate and the active agent to be transported into the target cells. Particularly in embodiments where the substrate is an antibody, the antibody is not monoclonal antibody 3E10 (or a fragment thereof). In some embodiments, the nucleoside transportation pathway includes either an equilibrative or concentrative nucleosidetransporter. In embodiments in which the nucleoside transport pathway involves an equilibrative nucleoside transporter, such a transporter may be insensitive to inhibition by low concentrations of nitrobenzylmercaptopurine riboside (NBMBR).”
“?Nucleoside transport pathways? These are systems that involve one or more transport proteins and affect the transport of a substrate over one or more biological membranes. A nucleoside transport pathway, for example, may mediate step-wise transporting a substrate across a plasma membrane and then the transport of the substrate through the intracellular membrane. The transport proteins and nucleoside translocators responsible for this step-wise translocation may be the same or different types of nucleoside transportationers. In some embodiments, the nucleoside transportationer might be an equilibrative nuclear transporter. Other embodiments may use a nucleoside transportationer that is a concentrative nucleoside carrier.
“A ?transport protein? or ?transporter? A protein that plays a role in the transport of a molecule across a membrane. This term can include membrane-bound proteins that recognize substrates and effect their entry into or exit from cells by receptor-mediated transporters or carrier-mediated transporters. Transporters can be found on intracellular organelles’ membranes and plasma membranes. Transporters are responsible for the movement of molecules into the intracellular organelles or the cytoplasm.
“Two distinct families of nucleoside transportationers (NTs), equilibrative and concentrative, have been identified. ?Equilibrative Nucleoside Transporters? ?Equilibrative nucleoside transporters? These transporters are those that move substrate down the concentration gradient by passive transport or facilitated diffusion. ENT activity is not dependent on a sodium ion gradient. transporters. ENTs are categorized into one of two subtypes based on sensitivity to inhibition by nitrobenzylmercaptopurine riboside (NBMBR). The ENT subtype (equilibrative and sensitive) is the most common. ), is inhibited with?1nM NBMPR. The other subtype, (equilibrative?insensitive?ei?), is unaffected. Low concentrations of NBMPR (e.g., 1?M) do not affect.
“Four members the ENT family were cloned. They are called ENT1, ENT2, ENT3, ENT4, and ENT4. Although all four transport adenosine, they differ in their ability to transport nucleosides and nucleobases. ENT1 is an example of an es subtype transporter. GenBank Accession No. 81375 and GenBank Accession No. 8375 are examples of polynucleotide sequences that encode human ENT1. GenBank Accession Number. The corresponding sequence of amino acids is AAC51103.1. ENT1 expression is widespread in rodent and human tissues. However, levels of expression vary from tissue to tissue. ENT1 has been shown to transport a variety of purine and pyrimidine nucleosides.
“ENT2 is an ei type transporter. GenBank Accession No. 89358 contains examples of polynucleotide sequences that encode human ENT2. GenBank Accession Number. AAC39526 is the sequence of amino acids. ENT2 can be found in a variety of tissues including the heart, brain, heart, placenta and thymus. ENT2-expressing cancer cells include certain types of renal tumor cells. Breast tumor cells. Colon cancer cells. Stomach cancer cells. Lung cancer cells. Leukemia cells. There are other types of ENT-2-expressing cancer cells that are well-known in the art. For example, Lu X et. al., Journal of Experimental Therapeutics and Oncology 2:100?212, 2002 and Pennycooke ME et. al. Biochemical and Biophysical Communication 208: 951-959 2001. ENT2 is expressed in high levels in skeletal muscles. ENT2 can also be found in the membrane of organelles, such as the nucleus. ENT2 has been shown to transport a variety of purine and pyrimidine nucleosides as well as nucleobases.
“ENT3 is an ei type transporter. GenBank Accession No. consists of exemplary polynucleotide sequences that encode human ENT3. GenBank Accession Number. The corresponding sequence of amino acids is NP_060814. ENT3 is expressed in many tissues and is also abundant in placenta. ENT3 is a predominant intracellular protein that co-localizes in cultured cells with lysosomal marker. ENT3 has been shown to transport a variety of purine and pyrimidine nucleosides.
NBMPR weakly inhibits ENT4. GenBank Accession No. demonstrates the polynucleotide sequences that encode human ENT4. GenBank Accession Number. The corresponding sequence of amino acids is AAH47592. ENT4 is a common amino acid sequence and is found in the brain, skeletal muscles, and heart. ENT4 is also found in the intestine, pancreas and liver. ENT4 has been shown to transport a variety of purine, pyrimidine nucleosides, and serotonin.
“?Concentrative Nucleoside Transporters?” CNTs are shorthand for Concentrative Nucleoside Transporters. CNTs refers to a group nucleoside transporters which transport nucleosides or nucleoside analogues via active transport. CNTs use sodium gradients that result from differences in intracellular and extracellular sodium concentrations. This concentration gradient allows for the uphill or concentrative transporting of substrate across biological membranes. The sodium concentration gradient across mammalian cell walls favors the movement of nucleoside and sodium into cells. CNTs can therefore be considered to be?Na+-dependent? transporters. Three cloned members are currently part of the CNT family. They differ in substrate selectivity, and their ratio of sodium to substrate.
CNT1 has been shown to transport both pyrimidine nucleosides and adenosine. The former in a high-affinity, small-capacity fashion. CNT1 transport occurs in a 1:1 ratio sodium-to-nucleoside. GenBank Accession No. GenBank Accession Number. The corresponding sequence of amino acids is AAB53839.1. CNT1 is expressed primarily in epithelial cells in tissues such as the small intestine and kidneys, liver, and many other regions of the brain.
CNT2 has been shown to transport both purine nucleosides and uridine. CNT2 transport occurs in a 1:1 ratio sodium-to-nucleoside. GenBank Accession No. 62109 contains examples of polynucleotide sequences that encode human CNT2. GenBank Accession Number. AAB88539 is the sequence of amino acids. CNT2 can be found in many human tissues, including the liver, kidneys, brain, heart, liver, kidneys, brain, small intestine, colon, and placenta.
CNT3 has been shown to be selective in transporting purine nucleosides and pyrimidine nucleosides as well as other nucleoside analogues. Transport via CNT3 takes place at a ratio 2:1 sodium-to nucleoside ratio. GenBank Accession No. 305210 and GenBank Accession No. 05220 are examples of polynucleotide sequences that encode human CNT3. GenBank Accession Number. AAG22551 is the sequence of amino acids. CNT3 can be found in tissues like the trachea and pancreas as well as bone marrow and mammary glands. It is also expressed in low levels in the intestine and liver, placenta and prostate.
“A ?conjugate? “Conjugate” is a molecule that contains a substrate and can be transported via a nucleoside transport route linked to an active agent. A nucleoside transporter is also possible to transport the conjugate.
“A ?substrate? “Substrate” is a compound that is easily absorbed into cells or organelles by a transport protein. Substrates possess specific kinetic parameters, such as Vmax or Km for a particular transporter. Vmax is the amount of substrate molecules transported per unit of time at the saturating substrate concentration. Km is the concentration at which the substrate can be transported at half the speed of Vmax. A transporter’s substrate should have a high Vmax value. For transporting low concentrations, a low Km value is preferred. A high Km value is desired for transporting high concentrations. Vmax is affected both by the intrinsic turnover rate of a transporter (molecules/transporter protein) and transporter density in plasma membrane that depends on expression level. The intrinsic capacity of a compound that will be transported by a specific transporter is often expressed as Vmax of the compound/Vmax a control compound that has been identified as a substrate.
“Substance that can be transported via a nucleoside transportation pathway” A molecule compound that is capable of being taken into a cell or organelle by a nucleoside transportation protein or nucleoside carrier. Substrates of invention conjugates can be known substrates for nucleoside transporters, or they may be identified using methods that are known in the art. Substrates can include a nucleoside or nucleobase as well as a nucleotide and an oligonucleotide.
“The term “nucleobase” is a synonym for purine or pyrimidine bases. “Nucleobase” refers to purine and pyrimidine bases. Adenine, Cytosine, Guanine, Uracil, and Thymine are some examples. Modified bases include nucleobases, which can include pseudouridines, dihydrouridines, inosines, ribothymidines, 7-methylguanosine(m7G), hypoxanthine and xanthine.
“The term “nucleoside” is a generic term that refers to a purine or pyrimidine base. A purine or pyrimidine base is covalently linked with a 5-carbon sugar (i.e. pentose). The nucleoside that is sugar-based is a nucleoside. When it is 2-deoxyribose the nucleoside will be a ribonucleoside. Exemplary nucleosides include cytidine, uridine, adenosine, guanosine, and thymidine, and the corresponding deoxyribonucleosides, which form the basis of the nucleotides that form DNA and RNA.”
“Nucleoside analog” is a term that refers to a nucleoside. “Nucleoside analog” is a term that refers to a nucleoside in the modification of either the base moiety or the sugar moiety. These analogs mimic natural nucleosides and can be used to replace a nucleoside in cellular functions. Nucleosides can be included in DNA and RNA to replace the nucleoside. Some nucleoside analogs can be incorporated to prevent further elongation during nucleic acid synthesis. Many nucleoside analogues are anti-viral and anti-cancer. Examples of nucleoside analogs include inosine, deoxyadenosine analogs such as didanosine (2?,3?-dideoxyinosine, ddI) and vidarabine (9-?-D-ribofuranosyladenine), deoxycytidine analogs such as cytarabine (cytosine arabinoside, emtricitabine, lamivudine (2?,3?-dideoxy-3?-thiacytidine, 3TC), and zalcitabine (2?-3?-dideoxycytidine, ddC), deoxyguanosine analogs such as abacavir, (deoxy-)thymidine analogs such as stavudine (2?-3?-didehydro-2?-3?-dideoxythymidine, d4T) and zidovudine (azidothymidine, or AZT), and deoxyuridine analogs such as idoxuridine and trifluridine.”
“Active agent” is the term used herein. A molecule that exerts a biological effect on a cell is an active agent. The active agent can be an inorganic molecule or nucleic acid. In some embodiments, it may also be an organic molecule. In some embodiments, the active ingredient is a polypeptide.
“In other embodiments, the active agent is selected from the group consisting of ?-glucosidase, ?-L-iduronidase, ?-galactosidase A, arylsulfatase, N-acetylgalactosamine-6-sulfatase or ?-galactosidase, iduronate 2-sulfatase, ceramidase, galactocerebrosidase, ?-glucuronidase, Heparan N-sulfatase, N-Acetyl-?-glucosaminidase, Acetyl CoA-?-glucosaminide N-acetyl transferase, N-acetyl-glucosamine-6 sulfatase, Galactose 6-sulfatase, Arylsulfatase A, B, or C, Arylsulfatase A Cerebroside, Ganglioside, Acid ?-galactosidase GMI Gaiglioside, Acid ?-galactosidase, Hexosaminidase A, Hexosaminidase B, ?-fucosidase, ?-N-Acetyl galactosaminidase, Glycoprotein Neuraminidase, Aspartylglucosamine amidase, Acid Lipase, Acid Ceramidase, Lysosomal Sphingomyelinase and other Sphingomyelinase. In certain embodiments, the active agent is dystrophin, components of dystrophin-glycoprotein complex, the laminin-?2 chain, fukutin-related protein, LARGE, fukutin, EMD, LMNA, DMPK, ZNF9, and PABPN1, Glycogen synthase, Glucose-6-phosphatase, Debranching enzyme, Transglucosidase, Myophosphorylase, Phosphorylase, Phosphofructokinase, Acid Maltase Deficiency, Carnitine Palmityl Transferase, Phosphoglycerate Kinase, or Phosphoglycerate Mutase, or a nucleic acid encoding any of said proteins.”
“In some embodiments, the substrate may be an antibody or a fragment thereof. The antibody or fragment thereof can bind nucleosides and nucleotides as well as nucleobases. It is possible that the substrate is not an antibody in certain embodiments.
“In some embodiments, the substrate portion may be a DNA binding autoantibody. An example of such DNA binding autoantibodies is an antibody with the binding specificity as the antibody produced by the hybridoma with ATCC accession number PTA2439, antibody 3E10 and variants and/or functional pieces thereof. FIG. shows the nucleotide sequences and amino acids for the variable region in the heavy chain of mAb3E10. 3. FIG. shows the nucleotide sequences and amino acids for the variable region in the light chains of Ab 3E10. 4. VkIII, the light chain that is designated as such, has the DNA binding ability for mAb 3E10. VkIII, the preferred light chain for 3E10, is used in the present invention.
Antibodies that penetrate living cells can be toxic or injurious, which may explain some of the autoimmune disorders. However, antibody mAb3E10 does not cause any harm to the cells it penetrates in tissue culture. Studies in vitro also showed that mAb3E10 and scFv scFv fragments can transport large proteins such as catalase into cells in tissue culture. Additionally, mAb3E10 and fragments thereof (e.g. Fv) should not cause significant inflammation in vivo, which could limit the therapeutic efficacy a biologically active molecule. The hybridoma 3E10 produced monoclonal antibody 3E10. It was placed on permanent deposit at the American Type Culture Collection, University Boulevard, Manassas (Va. 20110-2209), USA on August 31, 2000 according to the Budapest Treaty accession number PTA-2439. They are maintained and made accessible according to the Budapest Treaty. These strains are not allowed to be used to infringe on the patent rights granted by any government.
“Specific binding” is the term used herein. Specific binding refers to antibody binding of an antigen. The antibody binds to antigens with an affinity of 10-8 M, but preferably with an affinity that is less than 10-8 M. It also binds to antigens with a lower affinity than its affinity for binding non-specific antigens (e.g. BSA, casein) or closely related antigens. Alternately, the antibody could bind with an affinity of approximately 106 M-1, 107 M-1, 108 M-1, or 109M-1 or higher. It binds to predetermined antigens with an affinity (as defined by KA) at least 10 times higher and preferably at least 100 times higher than its affinity to binding to a nonspecific antigen (e.g. BSA, casein), other than the predetermined or closely related antigen. The KA and KD of the antibody variant or functional piece may be the same as the hybridoma antibody with ATCC accession number PTA2439. The mAb 3E10 antibody variant or functional piece may have the same KA and KD in certain embodiments.
“The term ‘kd?” is used herein. “The term?kd?” (sec?) is used herein to denote the dissociation rate constant for a specific antibody-antigen interaction. This value is also known as the “koff value.”
“The term ‘ka? “The term?ka? refers to the association rate constant for a specific antibody-antigen interaction. KA is the term. (M) is the association equilibrium constant for a specific antibody-antigen interaction.
“The term ‘KD? “The term?KD” is used herein to denote the dissociation equilibrium constant for a specific antibody-antigen interaction.
“Naturally occurring antibodies are generally tetramers that contain two heavy chains and two light chains. The proteolytic enzyme papain can be used to cleave antibodies, causing each of the heavy chains in the tetramers to break down, resulting in three subunits. The Fab fragments are the two units made up of a light and a heavy chain fragment that are approximately equal in mass to each other. fragments). The Fc fragment is the third unit. It consists of two equal segments from the heavy chain. Although the Fc fragment is not essential for antigen-antibody binding and is often ignored, it is crucial in the later processes that are involved in the elimination of antigen from the body.
“As used herein, the expression?functional fragments? of an antibody that have the binding specificity as the antibody as produced from the hybridoma with ATCC accession number PTA2439? A fragment that has the same cell penetration characteristics as mAb3E10 is called mAb 3E10. In certain embodiments, the conjugate is made from a functional fragment of an antibody that has the same binding specificity as the antibody produced by hybridoma with ATCC accession number PTA2439 or antibody mAb3E10. The functional fragment that is used in conjugate may be selected from the following groups: F(ab), Fab and F(ab). Fv fragments, and single-chain Fv (scFv), are some examples of functional fragments used in conjugates. In some embodiments, the functional fragment can be either an Fv fragment or an scFv fraction. One example is that the functional fragment must include at least the antigen binding portion of mAb3E10. Another example is the functional fragments, which are scFv fragments that include the variable region (VH) of the heavy chain and the variable region (VK), of the kappa lighter chain (MAb 3E10). The nucleic acid encoding the chains in mAb E310 is reversed and placed with the V? cDNA being placed 5? VH. To facilitate in vitro purification and histological localization, one or more tags, preferably peptides (e.g. myc or His6) may be added to a conjugate. Some embodiments add a Myc tag and a He6 tag to the C-terminus.
As those skilled in the art will recognize, altered antibodies (e.g. chimeric, humanized and CDR-grafted) are also possible. These include a combination of two polypeptide chains components of an antibody (e.g. one arm of an antibody including a heavy and a light chain or an Fab fragment containing a VL and a H domain, or an Fv fragment containing a VL and VH domains or an Fv fragment containing a linker), a single chain Fv domain that is linked to a domain via a linker) and a domain through a linker. These antibodies can also be made by chemical synthesis, hybridoma or recombinant techniques, such as those described in (Sambrook et. al., Molecular Cloning. 2nd Ed. (Cold Spring Harbor Laboratory 1989); incorporated by reference and Harlow and Lane (Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory 1987), which are incorporated by reference. You can use both anti-peptide or anti-conjugate antibody (see Bahouth et. al., Trends Pharmacol). Sci. Sci. Particularly, see FIGS. For specific sequences of nucleotide or amino acid sequences, see FIGS. 2A, 2B, and 3 of the illustrative invention mAb3E10.
“For example, antigen-binding sequences in the Fv variable area may be replaced with sequences that are equivalent to human Fv variables regions. Morrison et.al. (Science 229,1202-1207, 1985), and Oi et.al. provide general reviews of humanized, chimeric antibodies. (BioTechniques 4:214, 1986). These methods include expressing, manipulating, or isolating the nucleic acids sequences that encode immunoglobulin Fv variable areas from at least one of the heavy or light chains. Experts in the art are familiar with the sources of this nucleic acid. For example, it can be obtained from an antibody-producing hybridoma. The recombinant DNA that encodes the humanized, chimeric, antibody or a fragment thereof can then be used to clone into an appropriate expression vector. Alternatively, humanized antibodies can be made by CDR substitution U.S. Patent. No. 5,225,539; Jones (1986) Nature 321:552-525; Verhoeyan et al. 1988 Science 239; 1534; and Beidler (1988). J. Immunol. 141:4053-4060. In certain embodiments, the antibody in the conjugate is either a CDR-grafted or humanized form of the hybridoma accession number PTA2439. Other embodiments use a humanized form or CDR-grafted antibody mAb3E10. FIGS. 2A, 2B, and 3 show the CDR regions for the illustrative invention. 2A,2B, and 3 can contain amino acid substitutions such that 1, 2, 3, 4, 5, 7, 8, 9, or 10 different amino acids from those shown in figures. There may be anywhere from one to five amino acid differences in some cases.
“Also used herein, refers to variants of antibodies having the binding specificity as an antibody produced by hybridoma with ATCC accession number PTA2439. Variants that retain the same cell penetration characteristics as mAb3E10 and have the same binding specificity (e.g. improved ability of specific cells to be targeted, better ability to penetrate cell membranes, improved ability for localization to the cellular DNA and so forth) are included. These variants may include those in which one or more conservative substitutions have been introduced to the heavy chain, light chain, and/or constant region(s). The variant may have a light-chain with an amino acid sequence that is at least 80%, at minimum 90%, or at most 95% identical to the sequence in SEQ ID No:8. Other embodiments have a variant with a heavy chain that has an amino acid sequence of at least 80%, at least 90%, or at most 95% identical to that set forth in SEQID NO:6. The invention also includes antibodies encoded by nucleic acids sequences that can hybridize under strict conditions to the 3E10 variable sequence coding sequence (e.g. SEQID NO:5 or SEQID NO:7), or encode amino acid sequencings at least 80 percent, at least 90%, or at most 95% identical to those set forth in SEQID NO:6 and SEQID NO:8.
These variants include those in which one or more substitutions have been introduced to the heavy chain nucleotide sequencing, the light-chain nucleotide sequencing and/or the constant area(s) of an antibody. The variant may have a light chain with a nucleotide sequencing at least 80%, at least 90%, or at most 95% identical to that set forth in SEQID NO:7. Other embodiments have a variant with a heavy chain that has a nucleotide sequencing at least 80%, at least 90%, or at most 95% identical to that set forth in SEQID NO:5.
“An exemplary variant of the present invention that is intended to be used in the practice of this invention is an mAb3E10 VH variant. It involves a single modification of the aspartic acid residue at 31 to asparagine (i.e. mAb3E10-31). U.S. Pat. describes the preparation and subsequent variations, as well as a demonstration of cell penetration. No. 7,189,396. This particular mAb 3E10 variant of mAb is particularly well-suited for the delivery of biological molecules into kidney and brain cells. You may also use functional fragments or other 3E10 variants to target biologically active molecules. There are many variants of these functional fragments, provided they have substantially the same cell penetration characteristics after conjugation with a biologically active molecule.
“In other embodiments, it is possible to generate novel substrates that target a specific nucleoside transportationer.” These novel substrates can be created using molecular modeling or protein mimetic methods based on known substrate structures.
“Conjugates where the substrate and active agents are polypeptides (i.e. protein conjugates) can also be created to place the active ingredient at the carboxy or amino terminus of the substrate using well-known methods of recombinant genetics. These conjugates can also be expressed in a host as a fusion proteins. Alternately, the active agent and substrate can be chemically linked using a peptide bond. One or more tags, such as myc or His6 (SEQID NO:12), or one or several repeats of GGGGS (SEQID NO:11), could be used as the linker. The art also contains additional peptide linkers. A skilled artisan will know that the sequence of the linkers can be modified depending on the specific polypeptide being linked to the antibody.
“Vectors that are suitable for preparation of protein conjugates include those made from baculoviruses, phages, plasmids, phagemids, cosmids, fosmids, bacterial artificial DNA, viral DNA and Pl-based artificial.chromosomes, yeast plasmids, and yeast artificial.chromosome. You can choose from vaccinia or adenovirus, SV40, or pseudorabies to make the viral DNA vector. For the practice of the invention methods, suitable bacterial vectors include pQE70 and pQE60. The following eukaryotic vectors are suitable for the practice of the invention methods: pWLNEO. pSG5, PSVK3, pBPV. pMSG and pSVLSV40. For the practice of the invention methods, suitable eukaryotics include pWLNEO and pXTI.
“Those skilled in the art can choose a suitable regulatory area to be included in such a Vector, for example, from lacI, apt. lambda PR and PL, trp and CMV immediate early. HSV thymidine kinase. Early and late SV40. Retroviral LTR and mouse metallothionein I regulatory regions.
“Host cells are those cells that can express the vectors encoding the protein conjugates. They include a bacterial, eukaryotic, yeast, insect, and plant cell. For example, E. coli, Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SD, CHO, COS (e.g. All host cells suitable for the practice of invention methods are COS-7, or Bowes melanomas cells.
Methods of creating conjugates in which the substrate is polypeptides and the active agent is small molecules or drug compounds are possible using methods that are known to the art. For example, methods for attaching a drug or other small molecule pharmaceutical to protein include bifunctional chemical linkers such as N-succinimidyl (4-iodoacetyl)-aminobenzoate; sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate; 4-succinimidyl-oxycarbonyl-?-(2-pyridyldithio) toluene; sulfosuccinimidyl-6-[?-methyl-?-(pyridyldithiol)-toluamido]hexanoate; N-succinimidyl-3-(-2-pyridyldithio)-proprionate; succinimidyl-6-[3(-(-2-pyridyldithio)-proprionamido]hexanoate; sulfosuccinimidyl-6-[3(-(-2-pyridyldithio)-propionamido]hexanoate; 3-(2-pyridyldithio)-propionyl hydrazide, Ellman’s reagent, dichlorotriazinic acid, S-(2-thiopyridyl)-L-cysteine, and the like. U.S. Pat. also discloses bifunctional linking molecules. Nos. Nos.
“A further embodiment of the invention provides methods for screening a conjugate to be transported by a nucleoside transportation pathway. This method involves contacting a cell that expresses the nucleoside pathway with a conjugate; and determining if the conjugate is transported into the cells by the nucleoside pathway. The determining step may include comparing the amount conjugate transported into cells expressing nucleoside transportation system with the amount transported into control cells that do not express the nucleoside system. In these cases, an increase in the transport of conjugate from the cell expressing nucleoside pathway to the control cell is indicative of transport by that nucleoside pathway.
“In certain embodiments of the screening method, the nucleoside transportation pathway includes either an equilibrative or concentrative nucleosidetransporter. The equilibrative nuclear transporter may be selected from the following groups: ENT1, ENT2, ENT3, ENT4 and ENT5. In certain embodiments, the equilibrative nucleoside transporter is insensitive to low concentrations of nitrobenzylmercaptopurine riboside (NBMBR). Particular embodiments involve the transfection of the cell with DNA that encodes the nucleoside transportationer. These embodiments might also include an additional step where the amount conjugate transported into the cell transfected by DNA encoding nucleoside transportationer is compared with the amount conjugate transported into a cell untransfected with nucleosidetransporter. This is done to determine if transport is by nucleosidetransporter.
“Screening methods could also include compounds that inhibit nucleoside transportation activity. NBMPR, dilazep and dipyridamole are all inhibitors of certain es Nucleoside Transporters (e.g. ENT1). In certain embodiments, low levels of NBMPR might be used.
“In some embodiments, the conjugate might also contain a detectable label. These labels include radio-isotopes or fluorescent labels.
“Conjugates may be tested for their ability to be transported via nucleoside transport pathways. The screening takes place on cells that express the nucleoside pathway. Some methods involve transfection of cells with DNA that encodes a specific nucleoside transporter (NT). Other methods use cells expressing an endogenous NT. Endogenous CNTs or ENTs can be expressed by cells. An ENT may be the only NT that is expressed in some methods. Other methods use cells that express both ENT1 or ENT2 in their place.
Internalization of a compound can be detected by detecting signals from within cells from any one of many reporters. A reporter could be a simple label, such as a fluorophore or chromophore. If the spatial resolution of confocal imaging is sufficient to distinguish fluorescence from fluorescence within cells, it can also be used for internalization detection. Alternatively, confocal imaging can track the movements of compounds over time. Another method is to detect internalization using a reporter, which is a substrate of an enzyme within the cell. After the complex has been absorbed, the enzyme metabolizes it and emits either radioactive decay or an optical signal that indicates uptake. Commercial PMT-based imaging devices or CCD-based imaging system can monitor light emission. Additionally, there are other methods that use LCMS to detect the transported compounds and electrophysiological signals to indicate transport activity.
“In some cases, multiple conjugates can be screened simultaneously. The identity of each agent and conjugate moiety is tracked by labels attached to the conjugates. Some methods allow for screening to be done in competition mode. This means that a known substrate and a conjugate under testing are applied to the same cells. In such assays, the conjugate is usually differentially labeled with its known substrate. Alternately, the known substrate can be labeled so that parallel measurements of the uptake of the labeled substrate in the absence and presence test conjugate could be made.”
Comparing a conjugate’s Vmax with a known substrate can help to determine its relative value. A conjugate that has a minimum Vmax of 1%, preferably at 5%, more preferably 5%, more preferably 10%, most preferably at at least 20%, can be considered a substrate for the NT.
“Nucleic Acid Therapy”
“In some embodiments, the compositions may be used to deliver nucleic acid, or an analog thereof, to a targeted cell type or tissue. You can, for instance, reduce protein expression by using oligonucleotides, such as antisense, locked and peptide nucleic acid (LNA), Morpholinos, Morpholinos, and small interferingRNAs (siRNAs) from various chemistries. Expression constructs can also be delivered to cells to induce the expression of desired gene products.
“Nucleic acid that modulates the expression of a gene or gene product can be administered. A nucleic acid which modulates the expression of, as used in this article. . . ? This includes nucleic acid that regulates or down-regulates the expression of a given gene or gene product. An expression construct, for example, can be used to express the gene of your choice and cause up-regulation. A nucleic acid that causes downregulation could be, for instance, siRNA or a construct that encoding an antisenseRNA (such a short hairpinRNA) or a Ribozyme.
“Nucleic acids therapeutics, such oligonucleotides that target intracellular targets (mRNA and protein) are powerful therapeutic agents. Examples of oligonucleotide therapeutic agents include: antisense oligonucleotides, which are short, single-stranded DNAs and RNAs that bind to complementary mRNA and inhibit translation or induce RNaseH-mediated degradation of the transcript; siRNA oligonucleotides, which are short, double-stranded RNAs that activate the RNA interference (RNAi) pathway leading to mRNA degradation; ribozymes, which are oligonucleotide-based endonucleases that are designed to cleave specific mRNA transcripts; and nucleic acid aptamers and decoys, which are non-naturally occurring oligonucleotides that bind to and block protein targets in a manner analogous to small molecule drugs.”
“Nucleic Acid” is the term used herein. The term?nucleic acids” refers to polynucleotides, such as deoxyribonucleic and ribonucleic acids (RNA). As appropriate to the context and applicable to the described embodiment, the term should be understood to encompass both single-stranded and double-stranded nucleotides, such as antisense, What is the term “nucleic acid?”? The term “nucleic acid” can be used to describe DNA molecules, RNA molecules and siRNA molecules as well as native RNA molecules. It also includes DNA molecules in the form plasmids and cDNA as well as linear DNA, anti-sense DNA stands, oligos, or siRNA molecules. RNA molecules are also included (in the forms of siRNA, miRNA, shRNA, or ribozymes) proteins (antibodies or polypeptides), proteins, nucleic acid conjugated to other compounds (such small molecular inhibitors (SMI) of certain proteins) and fluorescent dyes. At this point, there are many nucleic-based therapeutic agents. Antisense agents, aptamers and ribozymes are just a few of the many types. M. Faria, H. Ulrich, Curr. Cancer Drug Targets 2002, 2 : 355-368
Antisense agents are the most advanced class, with one product (fomivirsen), on the market to treat CMV retinitis and another (alicaforsen), in advanced clinical trials for Crohn’s disease treatment. (oblimersen sodium), Oncomyc?, Affinitac? and Oncomyc? In clinical trials for the treatment of cancer. Antisense agents are usually short, chemically modified oligonucleotide chain that can hybridize to a specific area of a targeted target mRNA. The resulting mRNA doubleplex is degraded and recognized by RNAse H. This destroys the mRNA. The mRNA instructions are not able to reach the ribosome and prevent the production of the target mRNA protein. Antisense drugs are able to produce therapeutic benefits by inhibiting the production proteins that are involved in diseases.
An aptamer can be defined as a DNA orRNA molecule that has been chosen from a biased or random pool of oligonucleic acid molecules based on its ability or resistance to binding to a target molecule. There are many aptamers that can bind nucleic acid, proteins, small organic compound, or specific cell surfaces. Many have also been created to bind proteins associated with diseases. Aptamers can be made more quickly than antibodies and more readily chemically modified than antibodies. They are also more adaptable and more easily?evolved’. An iterative process that combines random modification and affinity-based screening allows for tighter binding to the target. These aptamers are expected to be useful in therapeutics and other applications where antibodies are not required. Macugen is at least one of the products. Advanced clinical trials are underway for pegaptanib sodium (a PEGylated Aptamer with high affinity to VEGF) in the treatment of age-related macular damage.
“Ribozymes or RNA enzymes are RNA molecules capable of catalyzing a chemical reaction. All known ribozymes have catalyzed the cleavage and synthesis of RNA. They come in a variety of sizes, including the large “hammerhead”. They range in size from the large?hammerhead? to the smaller?minizymes’. These are synthetic constructs that contain the minimum structures necessary for activity. Similar properties were also found in DNA-based enzymes (deoxyribozymes or DNAzymes). They have the potential to be used as therapeutic agents due to their ability to cut and recognize specific mRNA molecules. A ribozyme that catalyzes the cleavage a specific mRNA could be used as a therapeutic agent in much the same way as an antisense nucleic acids. However, a single ribozyme can destroy many copies. A synthetic ribozyme (Angiozyme?) It cleaves mRNA encoding a subtype of VEGF receptor. This is currently being studied in clinical trials to treat cancer.
“RNA interference (RNAi), is the phenomenon of gene specific post-transcriptional silencing using double-stranded RNA oligomers. Nature 2001, 411: 494-498; Caplen et al., Proc. Natl. Acad. Sci. U.S.A. 2001, 98: 9742-9747). Antisense oligonucleic acid and ribozymes have the potential to be therapeutic agents, reducing harmful protein expression by small inhibitoryRNAs (siRNAs). A protein complex, the RNA induced silencing compound, recognizes siRNA as double-stranded. This complex strips away one strand, facilitates hybridization with the target target mRNA and then cleaves that strand. For the same reason DNA-based vectors are capable of creating siRNA within cells. Short hairpin RNAs are also useful for this purpose. SiRNAs that can target specific genes, both endogenously or exogenously, have been described. See Paddison et al. Proc. Natl. Acad. Sci. U.S.A., 2002, 99: 1443-1448; Paddison et al., Genes & Dev. 2002, 16: 948-958; Sui et al. Proc. Natl. Acad. Sci. U.S.A. 2002. 8: 5515-5520. Brummelkamp and co., Science 2002. 296: 553-553.
“The term “nucleic acid-based therapeutic agents?” Three classes of compounds are included in the definition. This term includes all pharmaceutically acceptable salts and esters, prodrugs and codrugs as well as protected forms of the compounds, analogues and derivatives. The first class is collectively referred to as “antisense nucleic acid”. The first class, also known as?antisense nucleic acid, is composed of nucleic acids with 50 or less monomers that can be hybridized in a sequence-specific fashion to a single-stranded or targeted DNA molecule. This class includes ordinary DNA, RNA oligomers and DNA with modified backbones. It also includes phosphorothioates and methylphosphonates as well as peptide nucleic Acids, 2?-deoxy derivatives and nucleic Acid Oligomers that have chemically modified purine bases and pyrimidine bases. These oligomers can be lipophilically modified and/or pEGylated to alter their pharmacodynamics. This class also includes Oligomers that act as precursors to such agents, such hairpin RNAs, which are converted into siRNAs in cells.
Aptamers are the second type of nucleic acids-based therapeutic agents. Aptamers are nucleic acid oligomers with 50 or less monomer units that can bind with structural specificity to non-oligonucleotide target molecules or an oligonucleotide using sequence-specific hybridization. This class includes DNA and RNA Aptamers. Modifications of these nucleic acids include mirror-image DNA (?Spiegelmers) and RNA (?) ), peptide nucleic and nucleic acid Oligomers that have been chemically modified in any other way than what is described above. Any of these species can also contain chemically modified purines or pyrimidines, and may also be lipophilically modified (or PEGylated). M. Rimmele’s Chembiochem. 2003, 4: 963-71, and A. Vater, Curr. Opin. Drug Discov. Devel. 2003, 6: 253-61 to see recent reviews of aptamer tech. You will appreciate that members of the second class have, in addition to a structure-specific affinity for target molecules, a sequence-specific affinity towards a putative DNA sequence or RNA sequence.
“The third category of nucleic-acid-based therapeutic agents, also known as ‘nucleic acids enzymes?, is the third. This class includes nucleic acid-based therapeutic agents that can recognize and catalyze the cleavage target RNA molecules in a sequence-specific way. This class also includes hammerhead and minimized hammerheads (or minizymes). ), ?10-23? deoxyribozymes (?DNAzymes? ), and other similar. The class also includes chemically modified catalytic species, just like antisense molecules and aptamer compounds.
“Pharmaceutically acceptable salts” is a term that refers to the compounds of the invention. The term “pharmaceutically acceptable salts” refers to the physiologically and pharmaceutically acceptable salts for the compounds of the invention.
“A ?protein coding sequence? A sequence that?encodes? A sequence that?encodes? a specific polypeptide. A start codon at the 5 determines the boundaries of the coding sequence. (amino terminus) and a translation end codon at 3? (carboxyl) terminus. A coding sequence may include, but not be limited to, cDNA derived from prokaryotic and eukaryotic genomic mRNA, as well as cDNA sequences derived from prokaryotic and eukaryotic DNA. It can also include synthetic DNA sequences. Usually, a transcription termination sequence is located at 3? To the coding sequence.”
“The term ‘RNAi construct’ is used herein. It is a generic term that includes siRNA, hairpinRNA and other RNA species that can be cleaved live to make siRNAs. RNAi constructs also include expression vectors, also known as RNAi expression Vectors. These vectors can give rise to transcripts that form dsRNAs or RNAi in cells and/or transcripts that can be converted to siRNAs.
“As used in this document, the term “vector”? A nucleic acids molecule that can transport another nucleic acids to which it is linked. A genomic integrated vector (or?integrated vector) is one type of vector. These can be integrated into the host cell’s chromosomal genome. An episomal vector is another type of vector. It is a nucleic acids capable of extra-chromosomal reproduction. These vectors are known as expression vectors. In this specification, “plasmid” is defined as: If the context is not clear,?plasmid? und?vector are interchangeable. Unless otherwise stated, they are interchangeable. Expression vectors can contain regulatory elements that control transcription. These elements may be derived from insect, mammalian, or microbial genes. A host’s ability to reproduce is usually conferred by an origin. Additionally, a selection gene that facilitates recognition of transformants can be included. You can use vectors derived from viruses such as retroviruses and adenoviruses.
“The present disclosure is about the use of antisense nuclear acid to reduce the expression of a targeted disease-related proteins. An antisense nucleic acids can be delivered to cells, such as as expression plasmids that produce RNA that is complementary to the cellular portion of the mRNA that encodes the targeted disease-related proteins. The construct can also be an oligonucleotide, which is ex vivo generated and introduced into cells to inhibit expression. This is done by combining the mRNA or genomic sequences that encode the targeted disease-related proteins with it. These oligonucleotides can be optionally modified to resist endogenous exonucleases or endonucleases. Exemplary nucleic acids molecules that can be used as antisense Oligonucleotides include phosphoramidate and phosphothioate. (See U.S. Pat. Nos. 5,176,996 and 5,264,564, respectively. Van der Krol and colleagues (1988), Biotechniques 6; 958-976, and Stein et. al. (1988), Cancer Res 48: 2659?2668, have reviewed the general approaches for constructing oligomers that are useful in nucleic acids therapy.
“In other embodiments, the application relates to the use RNA interference (RNAi), to effect knockdown the targeted gene. RNAi constructs are double-stranded RNA which can be used to block the expression of a targeted gene. RNAi constructs may contain RNA that is identical or substantially the same as the target nucleic acids sequence or RNA that is identical or substantially the same as a specific region of the target sequence.
“Optionally, RNAi constructs can contain a nucleotide sequencing that hybridizes under physiologic circumstances of the cell with the nucleotide structure of at least a part of the mRNA transcript (the?target?) gene). Double-strandedRNA must be identical to natural RNA in order to be able to induce RNAi. The invention allows for embodiments that can tolerate sequence variations due to genetic mutations, polymorphic sites or evolutionary divergence. Although the number of nucleotide mismatches that can be tolerated between the target sequence sequence and the RNAi construct sequencing may reach 1 in 5, it is preferably less than 1 in 10. The most important mismatches in siRNA duplex’s center are the most serious and can essentially stop cleavage. Nucleotides located at the 3? Nucleotides at the 3? end of siRNA strands that are complementary to target RNA don’t significantly affect target recognition specificity. Sequence comparison and alignment algorithms are available to optimize sequence identity (see Gribskov & Devereux, Sequence Analysis Primer Stockton Press 1991 and references therein). The Smith-Waterman algorithm, which is implemented in the BESTFIT software program with default parameters (e.g. University of Wisconsin Genetic Computing Group), can be used to calculate the percent difference between nucleotide sequences. Preferably, between 90% and 100% sequence identity is maintained between the inhibitoryRNA and the target gene portion. Alternativly, the functional definition of the duplex region in RNA could be that it is a nucleotide sequence capable of hybridizing with target gene transcripts after hybridization at 50.degree for 12-16 hours. C. to 70.degree. C. in 400mM NaCl and 40mM pH 6.4 PIPES pH 6.4. Followed by washing with 1.0 mM EDA.
The double-stranded structure can be made by one self-complementary or two complementary RNAs strands. The dsRNA can be formed inside or outside the cell. You can introduce the RNA in a quantity that allows for delivery of at most one copy per cell. Double-stranded material can be administered in higher doses (e.g. at least 5, 10, 100 or 500 copies per cell), which may result in more effective inhibition. However, lower doses might prove useful for certain applications.
“The subject RNAi constructs may be?small-interfering RNAs?” Or?siRNAs. These nucleic acid are shorter than 50 and usually have a length of 19-30 nucleotides, but more preferably 21-23. SiRNAs recruit nuclease clusters and guide complexes to target mRNA by matching to specific sequences. The siRNAs in the protein complex degrade the target mRNA. A particular embodiment of siRNA molecules 21-23 includes a 3? The hydroxyl group. The siRNA constructs may be made by processing longer double-stranded RNAs in certain embodiments. One embodiment uses the Drosophila In vitro System. This embodiment combines dsRNA with a soluble extract derived directly from Drosophila embryos, thereby creating a combination. This combination is kept under conditions where the dsRNA can be processed to RNA molecules ranging from 21 to 23 nucleotides. There are many techniques that can be used to purify siRNA molecules, including gel electrophoresis. Non-denaturing methods such as column chromatography and size exclusion chromatography can also be used to purify siRNAs.
The chemical synthesizer or recombinant nucleic acids techniques can both be used to produce RNAi constructs. The cell treated may have an endogenous RNApolymerase that mediates transcription in vivo. Cloned RNApolymerase can also be used to produce transcription in vitro. Modifications to the phosphate sugar backbone or nucleoside may be made to RNAi constructs. These modifications can reduce susceptibility to cellular nuclear nucleases, increase bioavailability, improve formulation characteristics and/or alter other pharmacokinetic properties. The phosphodiester linkages in natural RNA can be modified to include at most one nitrogen or sulfur heteroatom.
Modifications to RNA structure can be tailored to permit specific genetic inhibition, while avoiding a broad response to dsRNA. Bases can also be modified to inhibit the activity of adenosine desminase. You can make the RNAi construct either enzymatically, or through partial/total organic synthesis. Any modified ribonucleotide may also be created by organic synthesis or in vitro enzymatic synthesis. You can modify RNAi constructs by chemically modifying RNA molecules (see Heidenreich et. al. (1997) Nucleic Acids Res. 25: 776-780; Wilson et al. (1994) J. Mol. Recog. 7: 89-98; Chen et al. (1995) Nucleic Acids Res. 23: 2661-2668; Hirschbein et al. (1997) Antisense Nucleic Acid Drug, Dev. 7: 55-61). For example, the backbone of an RNAi construct can be modified with phosphorothioates, phosphoramidate, phosphodithioates, chimeric methylphosphonate-phosphodiesters, peptide nucleic acids, 5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g., 2?-substituted or 2?-deoxy ribonucleosides, .alpha.-configurations, etc.).”
“In certain embodiments, at most one strand of siRNA molecules could have a 3? Overhangs can be as short as 1 to 6 nucleotides. The preferred number of overhangs is 3. overhangs are 1-3 nucleotides in length. One strand may have a 3? The overhang is different for each strand. One strand may be blunt-ended, while the other may have an overhang. Each strand may have different overhang lengths. The 3? can be used to enhance siRNA stability. Overhangs can be protected from degradation. One embodiment of the invention includes purine nucleotides such as adenosine and guanosine nucleotides to stabilize the RNA. Alternately, you can substitute pyrimidine nucleotides with modified analogues (e.g. substitution of uridine ucleotide 3). Overhangs caused by 2?-deoxythymidine may be tolerated, but this will not affect the effectiveness of the RNAi. In the absence of a 2? The absence of a 2?hydroxyl increases the nuclease resistance to the overhang in tissue culture medium and may also be beneficial in vivo.
“The RNAi construct may also come in the form a long, double-stranded RNA that is intracellularly digested to create a siRNA sequence. The RNAi construct could also be a hairpin RNA. The art of siRNA production is possible by processing hairpinRNAs in cells. Exogenous hairpin RNAs are possible to be synthesized or formed in vivo by transcribing RNA polymerase II promoters. For example, Paddison et. al. Genes Dev 2002, 16: 948-58, McCaffrey et. Nature 2002, 418, 38-9, McManus et., RNA 2002, 8: 842-50, Yu et. al. Proc. Natl. Acad. Sci. USA, 2002, 99: 6047-52). These hairpin RNAs can be engineered in cells, or in animals to ensure the continuous and stable suppression a desired gene.
“PCT Application WO 01/77350 describes an example vector for bidirectional transcription of a transcript to produce both sense and antisense transcripts from the same transgene within a eukaryotic cells. In certain embodiments, this invention provides a recombinant virus with the following characteristics: It comprises a viral replicon that has two overlapping transcription unit arranged in opposing orientations and flanking a transgene to produce an RNAi construct. The two overlapping transcription units are able to generate both sense and antisense transcripts from the same transgene in a host cells.
“In another embodiment, this disclosure refers to the use ribozyme compounds designed to catalytically clear an mRNA transcript to stop translation (see, for example, PCT International Publication W90/11364, Oct. 4, 1990; Sarver and al., 1990 Science 247: 1222-21225; and U.S. Patent. No. 5,093,246). Any ribozyme capable of cleaving the target mRNA at a site specific recognition sequence can be used, but hammerheads ribozymes are preferred. Hammerhead ribozymes cleave target mRNAs at specific locations that are dictated by flanking areas that form complementary base pair with the target mRNA. The only requirement is that the target’s mRNA contain the following sequence: 5?-UG-3?. It is well-known that hammerheads ribozymes can be constructed and produced. This is discussed in Haseloff & Gerlach 1988, Nature 334: 585-591. The ribozymes described in the present invention also include RNA Endoribonucleases (Cech-type ribozymes). The present invention also includes RNA endoribonucleases (?Cech-type ribozymes?) WO88/04300 from University Patents Inc. (Been and Cech 1986, Cell 47: 207-216)
“A further embodiment of the invention is the use DNA enzymes to inhibit the expression of a targeted genes. DNA enzymes combine some of the mechanistic characteristics of both antisense as ribozyme technology. DNA enzymes are designed to recognize a specific target sequence of nucleic acids, much like antisense oligonucleotides. However, they are catalytic and cleave that target nucleic acids much like ribozymes. In short, in order to create a DNA enzyme that recognizes and cleaves the target nucleic acids, someone skilled in the art must first identify the target sequence. The sequence should be a G/C rich, approximately 18-22 nucleotides. A high G/C content will ensure a stronger interaction between DNA enzyme and target sequence. The specific antisense sequence that will target DNA enzyme to the message is split so that it includes the two arms of DNA enzyme. The DNA enzyme loop is then placed between these two specific arms. U.S. Pat. describes methods for making and administering DNA enzymes. No. 6,110,462.”
The methods described herein can be used to deliver a variety molecules including, but not limited, small molecules (including those that are not optimally cell-permeability), lipids and nucleotides as well as nucleic acids, nucleotides polynucleotides polynucleotides polynucleotides polynucleotides polynucleiotides, nucleic acid, polynucleotides polynucleotides to polyamines, hormones, or polyamines to cross cellular membranes. Non-limiting examples of polynucleotides that can be delivered across cellular membranes using the compounds and methods of the invention include short interfering nucleic acid (siNA), antisense, enzymatic nucleic acid molecules, 2?,5?-oligoadenylate, triplex forming oligonucleotides, aptamers, and decoys. You can deliver biologically active molecules such as antibodies (e.g. monoclonal or chimeric, humanized, etc. ), cholesterol and hormones, antivirals and peptides.
The compounds, compositions and methods of invention can increase the delivery or availability biologically active molecules to cells and tissues (e.g. siNAs and siRNAs and siRNA inhibitors), compared with delivery of the molecules to cells or tissues in the absence of the compositions and methods. The presence of compounds and compositions from the invention can increase the biologically active molecules in cells, tissues, and organisms compared to when they are absent.
“The term ‘ligand? refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter that is capable of interfacing with another compound. “The term?ligand” refers to any compound or molecular, such a drug or peptide, hormone or neurotransmitter, that can interact with another compound such as a receptor either directly or indirectly. A receptor that interacts directly or indirectly with a ligand may be found on the cell’s surface. Alternately, it can also be an intracellular receptor. Interaction between the receptor and the ligand can lead to a biochemical response or a physical interaction. Galactose, galactosamine and N-acetylgalactosamine are all examples of ligands. A linker molecule can attach the ligand to a compound according to the invention. Examples include an amido, carbonyl; ester, protein, disulphide or silane, nucleosides, nucleosides, nucleosides, and nucleosides. One embodiment of the linker is a biodegradable one.
“Linkers”
A variety of linkers can be used to connect the substrate that is capable of being transported to active agent. You can use both degradable or cleavable links.
Summary for “Intranuclear Protein Transduction Through a Nucleoside Salvage Pathway”
“1. “1.
“The invention concerns conjugate molecules, and more specifically to conjugate molecule’s use in the delivery active agents to cells via endogenous cellular transportation pathways.
“2. Background Information”
“Transporter proteins play a role in the cellular uptake and/or passage of different molecules into and through cells. Carrier-mediated transport systems are proteins that are anchored to the cell’s membrane by multiple membrane-spanning domains. They function by transporting substrates through active and passive mechanisms. Carrier-mediated transport systems play an important role in the active and passive, facilitated transportation of important nutrients like vitamins, sugars, as well as amino acids. Organs like the liver and kidney also have carrier-mediated transporters. These proteins are involved with the excretion and re-absorption circulating compounds. The lipid bilayers of cellular membranes are often unable to diffuse hydrophilic or polar compounds. There are specific carriers-mediated transporters that allow active transport of small molecules, such as amino acids, di-, tripeptides, nucleosides, monosaccharides and water-soluble vitamins, across biological membranes.
“Nucleoside transporters are the primary carriers that mediate the uptake and release of physiological nucleosides, as well as many of their synthetic counterparts, by mammalian cells. There are two types of nucleoside transporters: (i), equilibrative (facilitated dispersion) and (ii), concentrative (secondary activate) sodium-dependent. Two equilibrative transport systems with similar broad substrate specificities have been identified and designated as the es (equilibrative sensitive) and ei (equilibrative insensitive) transporters, on the basis of their sensitivity or insensitivity to inhibition by nitrobenzylthioinosine (NBMPR, 1), respectively. Functionally, six sodium ion-coupled nucleoside transporters (concentrative) have been identified in mammalian tissues. They are cif/N1, cib/N3, cib/N3, cs/N5, cs/N6 and cs/N5.
“The anti-DNA antibody fragment3E10 Fv has been shown to be a novel molecular transport vehicle. It penetrates living cells with specific nuclear location, lack of toxicity, and delivers therapeutic cargo proteins in vitro as well as in vivo. To develop new molecular therapies that rely on intranuclear macromolecule transduction, it is crucial to understand the mechanism by which 3E10 Fv crosses cell membranes.
“The invention was based upon the discovery that a DNA binding antibody can penetrate cells and locate in the nucleus. It is transported via a nucleoside transport path.”
“Accordingly to one embodiment, conjugates are provided that include a substrate capable of being transported via a nucleoside transportation pathway and an active agent linked with the substrate. The conjugate is then transported using the nucleosidetransport pathway. Particularly in embodiments where the substrate is an antibody, the antibody is not monoclonal antibody 3E10 (or a fragment thereof). In some embodiments, the nucleoside transportation pathway includes either an equilibrative or concentrative nucleosidetransporter. In embodiments in which the nucleoside transport pathway involves an equilibrative nucleoside transporter, such a transporter may be insensitive to inhibition by low concentrations of nitrobenzylmercaptopurine riboside (NBMBR).”
“Another embodiment of the invention provides methods for delivering a conjugate into a target cell that expresses a nucleoside transportation pathway. These methods involve contacting a target cell that expresses the nucleoside transportation pathway with a conjugate, which includes a substrate capable of being transported via a nucleoside pathway and an active agent linked thereto. The conjugate is then transported through the nucleoside pathway. Particularly in embodiments where the substrate is an antibody, the antibody must not be the monoclonal antibody3E10 or fragment thereof. In some embodiments, the nucleoside transportation pathway includes either an equilibrative or concentrative nucleosidetransporter. In embodiments in which the nucleoside transport pathway involves an equilibrative nucleoside transporter, such a transporter may be insensitive to inhibition by low concentrations of nitrobenzylmercaptopurine riboside (NBMBR).”
“A further embodiment of the invention provides methods for screening conjugates for transport via a nucleoside transportation pathway. These methods involve contacting a cell that expresses the nucleoside transportation pathway with a conjugate in suitable conditions for transport; and determining if the conjugate has been transported into the cell via the nucleoside pathway. In some embodiments, the determining step involves comparing the amount conjugate transported into cells expressing the nucleoside transportation system with the amount transported into control cells that do not express the nucleosidetransport system. These embodiments indicate that transport is via the nucleoside pathway if there is an increase in conjugate transport from the cell expressing it.
“Another embodiment of the invention provides methods for treating a disease in a cell or tissue that expresses a nucleoside transportation pathway. This method involves administering to the patient with the disease or disorder a conjugate that includes a substrate capable of being transported via the nucleoside transportation pathway and an active agent for treatment. The nucleoside pathway also transports the conjugate. The active agent is delivered to the cells and tissues by the conjugate being transported there. The active agent is delivered to the affected cells if the disease or disorder involves the skeletal muscles in certain embodiments.
“According yet another embodiment, methods are available for treating a gene disorder. The method involves administering a conjugate to a patient with a genetic disease. A substrate is capable of being transported via an equilibrative nuclear transporter. Further, the active agent is linked and the conjugate is then transported by the equilibrative nuclear transporter. Cells then receive the active agent. The active agent may be a gene or protein that is missing in patients with the genetic disorder.
“In some other aspects, the disclosure offers a method for treating cancer. The method involves administering a conjugate to a patient with cancer. A substrate is capable of being transported via an equilibrative nuclear transporter. Further, the conjugate can be transported via the equilibrative nuclear transporter. This allows the conjugate to be transported into cancerous cells and delivers the active agent. The active agent may be a tumor suppressor gene, tumor suppressor protein or even a tumor suppressor gene.
“Accordingly to another embodiment of the invention, methods for treating an autoimmune condition include administering to a patient with the disorder a compound that blocks transport via a nucleoside transport route.”
“The present disclosure also includes a pharmaceutical composition that contains a conjugate as described herein, and an agent that promotes ENT2 transcription in a tissue. The agent that promotes ENT2 expression is, in some instances, an agent that treats or inhibits Hypoxia or that inhibits HIF-1. It could be hypoxic tissue such as a hypoxic tumour, a tissue that has insufficient blood supply, an ulcer, diabetic ulcer, poorly healing wounds, an ischemic region, an area that is ischemic after stroke or an area that is ischemic from cardiovascular disease. The agent that inhibits HIF-1 may be used in certain embodiments. A siRNA, an RNaseI construct, or a miRNA which reduces HIF-1? expression. The HIF-1 may be used in some embodiments. HIF-1 is a chemotherapeutic agent, including topotecan (NSC 644221), PX-478, 17-AAG or bevacizumab. In some embodiments, the agent that treats hypoxia or inhibits it is one that normalizes tumor vasculature or alters the redox status of a tissue. Hypoxia can be treated or inhibited by excess oxygen, TSC or almitrine.
“Furthermore is provided a treatment method for ENT-2 deficient tissues. It involves: a) administering an agent to promote ENT2 expression/activity and b) administering one the conjugates herein.
“Before the methods are described, it should be understood that this invention does not limit to specific compositions, methods, or experimental conditions. As such compositions and methods may vary. The terminology used in this document is intended to describe particular embodiments and is not meant to limit the invention’s scope.
“As used herein and in the appended claims, singular forms?a,?an, and??the” If the context requires otherwise, plural references should be included. For example, a reference to “the method” could be used. Includes one or more methods and/or steps described herein that will be apparent to persons skilled in art upon reading this disclosure.
“Unless otherwise stated, all technical terms and scientific terms used in this invention have the same meanings as those commonly understood by an ordinary skilled person of the art to which it belongs.”
“According to the present invention, conjugates are provided that contain a substrate capable of being transported via a nucleoside transportation pathway and an active agent linked with the substrate. The conjugate is then transported using the nucleoside pathway. This allows the conjugate and the active agent to be transported into the target cells. Particularly in embodiments where the substrate is an antibody, the antibody is not monoclonal antibody 3E10 (or a fragment thereof). In some embodiments, the nucleoside transportation pathway includes either an equilibrative or concentrative nucleosidetransporter. In embodiments in which the nucleoside transport pathway involves an equilibrative nucleoside transporter, such a transporter may be insensitive to inhibition by low concentrations of nitrobenzylmercaptopurine riboside (NBMBR).”
“?Nucleoside transport pathways? These are systems that involve one or more transport proteins and affect the transport of a substrate over one or more biological membranes. A nucleoside transport pathway, for example, may mediate step-wise transporting a substrate across a plasma membrane and then the transport of the substrate through the intracellular membrane. The transport proteins and nucleoside translocators responsible for this step-wise translocation may be the same or different types of nucleoside transportationers. In some embodiments, the nucleoside transportationer might be an equilibrative nuclear transporter. Other embodiments may use a nucleoside transportationer that is a concentrative nucleoside carrier.
“A ?transport protein? or ?transporter? A protein that plays a role in the transport of a molecule across a membrane. This term can include membrane-bound proteins that recognize substrates and effect their entry into or exit from cells by receptor-mediated transporters or carrier-mediated transporters. Transporters can be found on intracellular organelles’ membranes and plasma membranes. Transporters are responsible for the movement of molecules into the intracellular organelles or the cytoplasm.
“Two distinct families of nucleoside transportationers (NTs), equilibrative and concentrative, have been identified. ?Equilibrative Nucleoside Transporters? ?Equilibrative nucleoside transporters? These transporters are those that move substrate down the concentration gradient by passive transport or facilitated diffusion. ENT activity is not dependent on a sodium ion gradient. transporters. ENTs are categorized into one of two subtypes based on sensitivity to inhibition by nitrobenzylmercaptopurine riboside (NBMBR). The ENT subtype (equilibrative and sensitive) is the most common. ), is inhibited with?1nM NBMPR. The other subtype, (equilibrative?insensitive?ei?), is unaffected. Low concentrations of NBMPR (e.g., 1?M) do not affect.
“Four members the ENT family were cloned. They are called ENT1, ENT2, ENT3, ENT4, and ENT4. Although all four transport adenosine, they differ in their ability to transport nucleosides and nucleobases. ENT1 is an example of an es subtype transporter. GenBank Accession No. 81375 and GenBank Accession No. 8375 are examples of polynucleotide sequences that encode human ENT1. GenBank Accession Number. The corresponding sequence of amino acids is AAC51103.1. ENT1 expression is widespread in rodent and human tissues. However, levels of expression vary from tissue to tissue. ENT1 has been shown to transport a variety of purine and pyrimidine nucleosides.
“ENT2 is an ei type transporter. GenBank Accession No. 89358 contains examples of polynucleotide sequences that encode human ENT2. GenBank Accession Number. AAC39526 is the sequence of amino acids. ENT2 can be found in a variety of tissues including the heart, brain, heart, placenta and thymus. ENT2-expressing cancer cells include certain types of renal tumor cells. Breast tumor cells. Colon cancer cells. Stomach cancer cells. Lung cancer cells. Leukemia cells. There are other types of ENT-2-expressing cancer cells that are well-known in the art. For example, Lu X et. al., Journal of Experimental Therapeutics and Oncology 2:100?212, 2002 and Pennycooke ME et. al. Biochemical and Biophysical Communication 208: 951-959 2001. ENT2 is expressed in high levels in skeletal muscles. ENT2 can also be found in the membrane of organelles, such as the nucleus. ENT2 has been shown to transport a variety of purine and pyrimidine nucleosides as well as nucleobases.
“ENT3 is an ei type transporter. GenBank Accession No. consists of exemplary polynucleotide sequences that encode human ENT3. GenBank Accession Number. The corresponding sequence of amino acids is NP_060814. ENT3 is expressed in many tissues and is also abundant in placenta. ENT3 is a predominant intracellular protein that co-localizes in cultured cells with lysosomal marker. ENT3 has been shown to transport a variety of purine and pyrimidine nucleosides.
NBMPR weakly inhibits ENT4. GenBank Accession No. demonstrates the polynucleotide sequences that encode human ENT4. GenBank Accession Number. The corresponding sequence of amino acids is AAH47592. ENT4 is a common amino acid sequence and is found in the brain, skeletal muscles, and heart. ENT4 is also found in the intestine, pancreas and liver. ENT4 has been shown to transport a variety of purine, pyrimidine nucleosides, and serotonin.
“?Concentrative Nucleoside Transporters?” CNTs are shorthand for Concentrative Nucleoside Transporters. CNTs refers to a group nucleoside transporters which transport nucleosides or nucleoside analogues via active transport. CNTs use sodium gradients that result from differences in intracellular and extracellular sodium concentrations. This concentration gradient allows for the uphill or concentrative transporting of substrate across biological membranes. The sodium concentration gradient across mammalian cell walls favors the movement of nucleoside and sodium into cells. CNTs can therefore be considered to be?Na+-dependent? transporters. Three cloned members are currently part of the CNT family. They differ in substrate selectivity, and their ratio of sodium to substrate.
CNT1 has been shown to transport both pyrimidine nucleosides and adenosine. The former in a high-affinity, small-capacity fashion. CNT1 transport occurs in a 1:1 ratio sodium-to-nucleoside. GenBank Accession No. GenBank Accession Number. The corresponding sequence of amino acids is AAB53839.1. CNT1 is expressed primarily in epithelial cells in tissues such as the small intestine and kidneys, liver, and many other regions of the brain.
CNT2 has been shown to transport both purine nucleosides and uridine. CNT2 transport occurs in a 1:1 ratio sodium-to-nucleoside. GenBank Accession No. 62109 contains examples of polynucleotide sequences that encode human CNT2. GenBank Accession Number. AAB88539 is the sequence of amino acids. CNT2 can be found in many human tissues, including the liver, kidneys, brain, heart, liver, kidneys, brain, small intestine, colon, and placenta.
CNT3 has been shown to be selective in transporting purine nucleosides and pyrimidine nucleosides as well as other nucleoside analogues. Transport via CNT3 takes place at a ratio 2:1 sodium-to nucleoside ratio. GenBank Accession No. 305210 and GenBank Accession No. 05220 are examples of polynucleotide sequences that encode human CNT3. GenBank Accession Number. AAG22551 is the sequence of amino acids. CNT3 can be found in tissues like the trachea and pancreas as well as bone marrow and mammary glands. It is also expressed in low levels in the intestine and liver, placenta and prostate.
“A ?conjugate? “Conjugate” is a molecule that contains a substrate and can be transported via a nucleoside transport route linked to an active agent. A nucleoside transporter is also possible to transport the conjugate.
“A ?substrate? “Substrate” is a compound that is easily absorbed into cells or organelles by a transport protein. Substrates possess specific kinetic parameters, such as Vmax or Km for a particular transporter. Vmax is the amount of substrate molecules transported per unit of time at the saturating substrate concentration. Km is the concentration at which the substrate can be transported at half the speed of Vmax. A transporter’s substrate should have a high Vmax value. For transporting low concentrations, a low Km value is preferred. A high Km value is desired for transporting high concentrations. Vmax is affected both by the intrinsic turnover rate of a transporter (molecules/transporter protein) and transporter density in plasma membrane that depends on expression level. The intrinsic capacity of a compound that will be transported by a specific transporter is often expressed as Vmax of the compound/Vmax a control compound that has been identified as a substrate.
“Substance that can be transported via a nucleoside transportation pathway” A molecule compound that is capable of being taken into a cell or organelle by a nucleoside transportation protein or nucleoside carrier. Substrates of invention conjugates can be known substrates for nucleoside transporters, or they may be identified using methods that are known in the art. Substrates can include a nucleoside or nucleobase as well as a nucleotide and an oligonucleotide.
“The term “nucleobase” is a synonym for purine or pyrimidine bases. “Nucleobase” refers to purine and pyrimidine bases. Adenine, Cytosine, Guanine, Uracil, and Thymine are some examples. Modified bases include nucleobases, which can include pseudouridines, dihydrouridines, inosines, ribothymidines, 7-methylguanosine(m7G), hypoxanthine and xanthine.
“The term “nucleoside” is a generic term that refers to a purine or pyrimidine base. A purine or pyrimidine base is covalently linked with a 5-carbon sugar (i.e. pentose). The nucleoside that is sugar-based is a nucleoside. When it is 2-deoxyribose the nucleoside will be a ribonucleoside. Exemplary nucleosides include cytidine, uridine, adenosine, guanosine, and thymidine, and the corresponding deoxyribonucleosides, which form the basis of the nucleotides that form DNA and RNA.”
“Nucleoside analog” is a term that refers to a nucleoside. “Nucleoside analog” is a term that refers to a nucleoside in the modification of either the base moiety or the sugar moiety. These analogs mimic natural nucleosides and can be used to replace a nucleoside in cellular functions. Nucleosides can be included in DNA and RNA to replace the nucleoside. Some nucleoside analogs can be incorporated to prevent further elongation during nucleic acid synthesis. Many nucleoside analogues are anti-viral and anti-cancer. Examples of nucleoside analogs include inosine, deoxyadenosine analogs such as didanosine (2?,3?-dideoxyinosine, ddI) and vidarabine (9-?-D-ribofuranosyladenine), deoxycytidine analogs such as cytarabine (cytosine arabinoside, emtricitabine, lamivudine (2?,3?-dideoxy-3?-thiacytidine, 3TC), and zalcitabine (2?-3?-dideoxycytidine, ddC), deoxyguanosine analogs such as abacavir, (deoxy-)thymidine analogs such as stavudine (2?-3?-didehydro-2?-3?-dideoxythymidine, d4T) and zidovudine (azidothymidine, or AZT), and deoxyuridine analogs such as idoxuridine and trifluridine.”
“Active agent” is the term used herein. A molecule that exerts a biological effect on a cell is an active agent. The active agent can be an inorganic molecule or nucleic acid. In some embodiments, it may also be an organic molecule. In some embodiments, the active ingredient is a polypeptide.
“In other embodiments, the active agent is selected from the group consisting of ?-glucosidase, ?-L-iduronidase, ?-galactosidase A, arylsulfatase, N-acetylgalactosamine-6-sulfatase or ?-galactosidase, iduronate 2-sulfatase, ceramidase, galactocerebrosidase, ?-glucuronidase, Heparan N-sulfatase, N-Acetyl-?-glucosaminidase, Acetyl CoA-?-glucosaminide N-acetyl transferase, N-acetyl-glucosamine-6 sulfatase, Galactose 6-sulfatase, Arylsulfatase A, B, or C, Arylsulfatase A Cerebroside, Ganglioside, Acid ?-galactosidase GMI Gaiglioside, Acid ?-galactosidase, Hexosaminidase A, Hexosaminidase B, ?-fucosidase, ?-N-Acetyl galactosaminidase, Glycoprotein Neuraminidase, Aspartylglucosamine amidase, Acid Lipase, Acid Ceramidase, Lysosomal Sphingomyelinase and other Sphingomyelinase. In certain embodiments, the active agent is dystrophin, components of dystrophin-glycoprotein complex, the laminin-?2 chain, fukutin-related protein, LARGE, fukutin, EMD, LMNA, DMPK, ZNF9, and PABPN1, Glycogen synthase, Glucose-6-phosphatase, Debranching enzyme, Transglucosidase, Myophosphorylase, Phosphorylase, Phosphofructokinase, Acid Maltase Deficiency, Carnitine Palmityl Transferase, Phosphoglycerate Kinase, or Phosphoglycerate Mutase, or a nucleic acid encoding any of said proteins.”
“In some embodiments, the substrate may be an antibody or a fragment thereof. The antibody or fragment thereof can bind nucleosides and nucleotides as well as nucleobases. It is possible that the substrate is not an antibody in certain embodiments.
“In some embodiments, the substrate portion may be a DNA binding autoantibody. An example of such DNA binding autoantibodies is an antibody with the binding specificity as the antibody produced by the hybridoma with ATCC accession number PTA2439, antibody 3E10 and variants and/or functional pieces thereof. FIG. shows the nucleotide sequences and amino acids for the variable region in the heavy chain of mAb3E10. 3. FIG. shows the nucleotide sequences and amino acids for the variable region in the light chains of Ab 3E10. 4. VkIII, the light chain that is designated as such, has the DNA binding ability for mAb 3E10. VkIII, the preferred light chain for 3E10, is used in the present invention.
Antibodies that penetrate living cells can be toxic or injurious, which may explain some of the autoimmune disorders. However, antibody mAb3E10 does not cause any harm to the cells it penetrates in tissue culture. Studies in vitro also showed that mAb3E10 and scFv scFv fragments can transport large proteins such as catalase into cells in tissue culture. Additionally, mAb3E10 and fragments thereof (e.g. Fv) should not cause significant inflammation in vivo, which could limit the therapeutic efficacy a biologically active molecule. The hybridoma 3E10 produced monoclonal antibody 3E10. It was placed on permanent deposit at the American Type Culture Collection, University Boulevard, Manassas (Va. 20110-2209), USA on August 31, 2000 according to the Budapest Treaty accession number PTA-2439. They are maintained and made accessible according to the Budapest Treaty. These strains are not allowed to be used to infringe on the patent rights granted by any government.
“Specific binding” is the term used herein. Specific binding refers to antibody binding of an antigen. The antibody binds to antigens with an affinity of 10-8 M, but preferably with an affinity that is less than 10-8 M. It also binds to antigens with a lower affinity than its affinity for binding non-specific antigens (e.g. BSA, casein) or closely related antigens. Alternately, the antibody could bind with an affinity of approximately 106 M-1, 107 M-1, 108 M-1, or 109M-1 or higher. It binds to predetermined antigens with an affinity (as defined by KA) at least 10 times higher and preferably at least 100 times higher than its affinity to binding to a nonspecific antigen (e.g. BSA, casein), other than the predetermined or closely related antigen. The KA and KD of the antibody variant or functional piece may be the same as the hybridoma antibody with ATCC accession number PTA2439. The mAb 3E10 antibody variant or functional piece may have the same KA and KD in certain embodiments.
“The term ‘kd?” is used herein. “The term?kd?” (sec?) is used herein to denote the dissociation rate constant for a specific antibody-antigen interaction. This value is also known as the “koff value.”
“The term ‘ka? “The term?ka? refers to the association rate constant for a specific antibody-antigen interaction. KA is the term. (M) is the association equilibrium constant for a specific antibody-antigen interaction.
“The term ‘KD? “The term?KD” is used herein to denote the dissociation equilibrium constant for a specific antibody-antigen interaction.
“Naturally occurring antibodies are generally tetramers that contain two heavy chains and two light chains. The proteolytic enzyme papain can be used to cleave antibodies, causing each of the heavy chains in the tetramers to break down, resulting in three subunits. The Fab fragments are the two units made up of a light and a heavy chain fragment that are approximately equal in mass to each other. fragments). The Fc fragment is the third unit. It consists of two equal segments from the heavy chain. Although the Fc fragment is not essential for antigen-antibody binding and is often ignored, it is crucial in the later processes that are involved in the elimination of antigen from the body.
“As used herein, the expression?functional fragments? of an antibody that have the binding specificity as the antibody as produced from the hybridoma with ATCC accession number PTA2439? A fragment that has the same cell penetration characteristics as mAb3E10 is called mAb 3E10. In certain embodiments, the conjugate is made from a functional fragment of an antibody that has the same binding specificity as the antibody produced by hybridoma with ATCC accession number PTA2439 or antibody mAb3E10. The functional fragment that is used in conjugate may be selected from the following groups: F(ab), Fab and F(ab). Fv fragments, and single-chain Fv (scFv), are some examples of functional fragments used in conjugates. In some embodiments, the functional fragment can be either an Fv fragment or an scFv fraction. One example is that the functional fragment must include at least the antigen binding portion of mAb3E10. Another example is the functional fragments, which are scFv fragments that include the variable region (VH) of the heavy chain and the variable region (VK), of the kappa lighter chain (MAb 3E10). The nucleic acid encoding the chains in mAb E310 is reversed and placed with the V? cDNA being placed 5? VH. To facilitate in vitro purification and histological localization, one or more tags, preferably peptides (e.g. myc or His6) may be added to a conjugate. Some embodiments add a Myc tag and a He6 tag to the C-terminus.
As those skilled in the art will recognize, altered antibodies (e.g. chimeric, humanized and CDR-grafted) are also possible. These include a combination of two polypeptide chains components of an antibody (e.g. one arm of an antibody including a heavy and a light chain or an Fab fragment containing a VL and a H domain, or an Fv fragment containing a VL and VH domains or an Fv fragment containing a linker), a single chain Fv domain that is linked to a domain via a linker) and a domain through a linker. These antibodies can also be made by chemical synthesis, hybridoma or recombinant techniques, such as those described in (Sambrook et. al., Molecular Cloning. 2nd Ed. (Cold Spring Harbor Laboratory 1989); incorporated by reference and Harlow and Lane (Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory 1987), which are incorporated by reference. You can use both anti-peptide or anti-conjugate antibody (see Bahouth et. al., Trends Pharmacol). Sci. Sci. Particularly, see FIGS. For specific sequences of nucleotide or amino acid sequences, see FIGS. 2A, 2B, and 3 of the illustrative invention mAb3E10.
“For example, antigen-binding sequences in the Fv variable area may be replaced with sequences that are equivalent to human Fv variables regions. Morrison et.al. (Science 229,1202-1207, 1985), and Oi et.al. provide general reviews of humanized, chimeric antibodies. (BioTechniques 4:214, 1986). These methods include expressing, manipulating, or isolating the nucleic acids sequences that encode immunoglobulin Fv variable areas from at least one of the heavy or light chains. Experts in the art are familiar with the sources of this nucleic acid. For example, it can be obtained from an antibody-producing hybridoma. The recombinant DNA that encodes the humanized, chimeric, antibody or a fragment thereof can then be used to clone into an appropriate expression vector. Alternatively, humanized antibodies can be made by CDR substitution U.S. Patent. No. 5,225,539; Jones (1986) Nature 321:552-525; Verhoeyan et al. 1988 Science 239; 1534; and Beidler (1988). J. Immunol. 141:4053-4060. In certain embodiments, the antibody in the conjugate is either a CDR-grafted or humanized form of the hybridoma accession number PTA2439. Other embodiments use a humanized form or CDR-grafted antibody mAb3E10. FIGS. 2A, 2B, and 3 show the CDR regions for the illustrative invention. 2A,2B, and 3 can contain amino acid substitutions such that 1, 2, 3, 4, 5, 7, 8, 9, or 10 different amino acids from those shown in figures. There may be anywhere from one to five amino acid differences in some cases.
“Also used herein, refers to variants of antibodies having the binding specificity as an antibody produced by hybridoma with ATCC accession number PTA2439. Variants that retain the same cell penetration characteristics as mAb3E10 and have the same binding specificity (e.g. improved ability of specific cells to be targeted, better ability to penetrate cell membranes, improved ability for localization to the cellular DNA and so forth) are included. These variants may include those in which one or more conservative substitutions have been introduced to the heavy chain, light chain, and/or constant region(s). The variant may have a light-chain with an amino acid sequence that is at least 80%, at minimum 90%, or at most 95% identical to the sequence in SEQ ID No:8. Other embodiments have a variant with a heavy chain that has an amino acid sequence of at least 80%, at least 90%, or at most 95% identical to that set forth in SEQID NO:6. The invention also includes antibodies encoded by nucleic acids sequences that can hybridize under strict conditions to the 3E10 variable sequence coding sequence (e.g. SEQID NO:5 or SEQID NO:7), or encode amino acid sequencings at least 80 percent, at least 90%, or at most 95% identical to those set forth in SEQID NO:6 and SEQID NO:8.
These variants include those in which one or more substitutions have been introduced to the heavy chain nucleotide sequencing, the light-chain nucleotide sequencing and/or the constant area(s) of an antibody. The variant may have a light chain with a nucleotide sequencing at least 80%, at least 90%, or at most 95% identical to that set forth in SEQID NO:7. Other embodiments have a variant with a heavy chain that has a nucleotide sequencing at least 80%, at least 90%, or at most 95% identical to that set forth in SEQID NO:5.
“An exemplary variant of the present invention that is intended to be used in the practice of this invention is an mAb3E10 VH variant. It involves a single modification of the aspartic acid residue at 31 to asparagine (i.e. mAb3E10-31). U.S. Pat. describes the preparation and subsequent variations, as well as a demonstration of cell penetration. No. 7,189,396. This particular mAb 3E10 variant of mAb is particularly well-suited for the delivery of biological molecules into kidney and brain cells. You may also use functional fragments or other 3E10 variants to target biologically active molecules. There are many variants of these functional fragments, provided they have substantially the same cell penetration characteristics after conjugation with a biologically active molecule.
“In other embodiments, it is possible to generate novel substrates that target a specific nucleoside transportationer.” These novel substrates can be created using molecular modeling or protein mimetic methods based on known substrate structures.
“Conjugates where the substrate and active agents are polypeptides (i.e. protein conjugates) can also be created to place the active ingredient at the carboxy or amino terminus of the substrate using well-known methods of recombinant genetics. These conjugates can also be expressed in a host as a fusion proteins. Alternately, the active agent and substrate can be chemically linked using a peptide bond. One or more tags, such as myc or His6 (SEQID NO:12), or one or several repeats of GGGGS (SEQID NO:11), could be used as the linker. The art also contains additional peptide linkers. A skilled artisan will know that the sequence of the linkers can be modified depending on the specific polypeptide being linked to the antibody.
“Vectors that are suitable for preparation of protein conjugates include those made from baculoviruses, phages, plasmids, phagemids, cosmids, fosmids, bacterial artificial DNA, viral DNA and Pl-based artificial.chromosomes, yeast plasmids, and yeast artificial.chromosome. You can choose from vaccinia or adenovirus, SV40, or pseudorabies to make the viral DNA vector. For the practice of the invention methods, suitable bacterial vectors include pQE70 and pQE60. The following eukaryotic vectors are suitable for the practice of the invention methods: pWLNEO. pSG5, PSVK3, pBPV. pMSG and pSVLSV40. For the practice of the invention methods, suitable eukaryotics include pWLNEO and pXTI.
“Those skilled in the art can choose a suitable regulatory area to be included in such a Vector, for example, from lacI, apt. lambda PR and PL, trp and CMV immediate early. HSV thymidine kinase. Early and late SV40. Retroviral LTR and mouse metallothionein I regulatory regions.
“Host cells are those cells that can express the vectors encoding the protein conjugates. They include a bacterial, eukaryotic, yeast, insect, and plant cell. For example, E. coli, Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SD, CHO, COS (e.g. All host cells suitable for the practice of invention methods are COS-7, or Bowes melanomas cells.
Methods of creating conjugates in which the substrate is polypeptides and the active agent is small molecules or drug compounds are possible using methods that are known to the art. For example, methods for attaching a drug or other small molecule pharmaceutical to protein include bifunctional chemical linkers such as N-succinimidyl (4-iodoacetyl)-aminobenzoate; sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate; 4-succinimidyl-oxycarbonyl-?-(2-pyridyldithio) toluene; sulfosuccinimidyl-6-[?-methyl-?-(pyridyldithiol)-toluamido]hexanoate; N-succinimidyl-3-(-2-pyridyldithio)-proprionate; succinimidyl-6-[3(-(-2-pyridyldithio)-proprionamido]hexanoate; sulfosuccinimidyl-6-[3(-(-2-pyridyldithio)-propionamido]hexanoate; 3-(2-pyridyldithio)-propionyl hydrazide, Ellman’s reagent, dichlorotriazinic acid, S-(2-thiopyridyl)-L-cysteine, and the like. U.S. Pat. also discloses bifunctional linking molecules. Nos. Nos.
“A further embodiment of the invention provides methods for screening a conjugate to be transported by a nucleoside transportation pathway. This method involves contacting a cell that expresses the nucleoside pathway with a conjugate; and determining if the conjugate is transported into the cells by the nucleoside pathway. The determining step may include comparing the amount conjugate transported into cells expressing nucleoside transportation system with the amount transported into control cells that do not express the nucleoside system. In these cases, an increase in the transport of conjugate from the cell expressing nucleoside pathway to the control cell is indicative of transport by that nucleoside pathway.
“In certain embodiments of the screening method, the nucleoside transportation pathway includes either an equilibrative or concentrative nucleosidetransporter. The equilibrative nuclear transporter may be selected from the following groups: ENT1, ENT2, ENT3, ENT4 and ENT5. In certain embodiments, the equilibrative nucleoside transporter is insensitive to low concentrations of nitrobenzylmercaptopurine riboside (NBMBR). Particular embodiments involve the transfection of the cell with DNA that encodes the nucleoside transportationer. These embodiments might also include an additional step where the amount conjugate transported into the cell transfected by DNA encoding nucleoside transportationer is compared with the amount conjugate transported into a cell untransfected with nucleosidetransporter. This is done to determine if transport is by nucleosidetransporter.
“Screening methods could also include compounds that inhibit nucleoside transportation activity. NBMPR, dilazep and dipyridamole are all inhibitors of certain es Nucleoside Transporters (e.g. ENT1). In certain embodiments, low levels of NBMPR might be used.
“In some embodiments, the conjugate might also contain a detectable label. These labels include radio-isotopes or fluorescent labels.
“Conjugates may be tested for their ability to be transported via nucleoside transport pathways. The screening takes place on cells that express the nucleoside pathway. Some methods involve transfection of cells with DNA that encodes a specific nucleoside transporter (NT). Other methods use cells expressing an endogenous NT. Endogenous CNTs or ENTs can be expressed by cells. An ENT may be the only NT that is expressed in some methods. Other methods use cells that express both ENT1 or ENT2 in their place.
Internalization of a compound can be detected by detecting signals from within cells from any one of many reporters. A reporter could be a simple label, such as a fluorophore or chromophore. If the spatial resolution of confocal imaging is sufficient to distinguish fluorescence from fluorescence within cells, it can also be used for internalization detection. Alternatively, confocal imaging can track the movements of compounds over time. Another method is to detect internalization using a reporter, which is a substrate of an enzyme within the cell. After the complex has been absorbed, the enzyme metabolizes it and emits either radioactive decay or an optical signal that indicates uptake. Commercial PMT-based imaging devices or CCD-based imaging system can monitor light emission. Additionally, there are other methods that use LCMS to detect the transported compounds and electrophysiological signals to indicate transport activity.
“In some cases, multiple conjugates can be screened simultaneously. The identity of each agent and conjugate moiety is tracked by labels attached to the conjugates. Some methods allow for screening to be done in competition mode. This means that a known substrate and a conjugate under testing are applied to the same cells. In such assays, the conjugate is usually differentially labeled with its known substrate. Alternately, the known substrate can be labeled so that parallel measurements of the uptake of the labeled substrate in the absence and presence test conjugate could be made.”
Comparing a conjugate’s Vmax with a known substrate can help to determine its relative value. A conjugate that has a minimum Vmax of 1%, preferably at 5%, more preferably 5%, more preferably 10%, most preferably at at least 20%, can be considered a substrate for the NT.
“Nucleic Acid Therapy”
“In some embodiments, the compositions may be used to deliver nucleic acid, or an analog thereof, to a targeted cell type or tissue. You can, for instance, reduce protein expression by using oligonucleotides, such as antisense, locked and peptide nucleic acid (LNA), Morpholinos, Morpholinos, and small interferingRNAs (siRNAs) from various chemistries. Expression constructs can also be delivered to cells to induce the expression of desired gene products.
“Nucleic acid that modulates the expression of a gene or gene product can be administered. A nucleic acid which modulates the expression of, as used in this article. . . ? This includes nucleic acid that regulates or down-regulates the expression of a given gene or gene product. An expression construct, for example, can be used to express the gene of your choice and cause up-regulation. A nucleic acid that causes downregulation could be, for instance, siRNA or a construct that encoding an antisenseRNA (such a short hairpinRNA) or a Ribozyme.
“Nucleic acids therapeutics, such oligonucleotides that target intracellular targets (mRNA and protein) are powerful therapeutic agents. Examples of oligonucleotide therapeutic agents include: antisense oligonucleotides, which are short, single-stranded DNAs and RNAs that bind to complementary mRNA and inhibit translation or induce RNaseH-mediated degradation of the transcript; siRNA oligonucleotides, which are short, double-stranded RNAs that activate the RNA interference (RNAi) pathway leading to mRNA degradation; ribozymes, which are oligonucleotide-based endonucleases that are designed to cleave specific mRNA transcripts; and nucleic acid aptamers and decoys, which are non-naturally occurring oligonucleotides that bind to and block protein targets in a manner analogous to small molecule drugs.”
“Nucleic Acid” is the term used herein. The term?nucleic acids” refers to polynucleotides, such as deoxyribonucleic and ribonucleic acids (RNA). As appropriate to the context and applicable to the described embodiment, the term should be understood to encompass both single-stranded and double-stranded nucleotides, such as antisense, What is the term “nucleic acid?”? The term “nucleic acid” can be used to describe DNA molecules, RNA molecules and siRNA molecules as well as native RNA molecules. It also includes DNA molecules in the form plasmids and cDNA as well as linear DNA, anti-sense DNA stands, oligos, or siRNA molecules. RNA molecules are also included (in the forms of siRNA, miRNA, shRNA, or ribozymes) proteins (antibodies or polypeptides), proteins, nucleic acid conjugated to other compounds (such small molecular inhibitors (SMI) of certain proteins) and fluorescent dyes. At this point, there are many nucleic-based therapeutic agents. Antisense agents, aptamers and ribozymes are just a few of the many types. M. Faria, H. Ulrich, Curr. Cancer Drug Targets 2002, 2 : 355-368
Antisense agents are the most advanced class, with one product (fomivirsen), on the market to treat CMV retinitis and another (alicaforsen), in advanced clinical trials for Crohn’s disease treatment. (oblimersen sodium), Oncomyc?, Affinitac? and Oncomyc? In clinical trials for the treatment of cancer. Antisense agents are usually short, chemically modified oligonucleotide chain that can hybridize to a specific area of a targeted target mRNA. The resulting mRNA doubleplex is degraded and recognized by RNAse H. This destroys the mRNA. The mRNA instructions are not able to reach the ribosome and prevent the production of the target mRNA protein. Antisense drugs are able to produce therapeutic benefits by inhibiting the production proteins that are involved in diseases.
An aptamer can be defined as a DNA orRNA molecule that has been chosen from a biased or random pool of oligonucleic acid molecules based on its ability or resistance to binding to a target molecule. There are many aptamers that can bind nucleic acid, proteins, small organic compound, or specific cell surfaces. Many have also been created to bind proteins associated with diseases. Aptamers can be made more quickly than antibodies and more readily chemically modified than antibodies. They are also more adaptable and more easily?evolved’. An iterative process that combines random modification and affinity-based screening allows for tighter binding to the target. These aptamers are expected to be useful in therapeutics and other applications where antibodies are not required. Macugen is at least one of the products. Advanced clinical trials are underway for pegaptanib sodium (a PEGylated Aptamer with high affinity to VEGF) in the treatment of age-related macular damage.
“Ribozymes or RNA enzymes are RNA molecules capable of catalyzing a chemical reaction. All known ribozymes have catalyzed the cleavage and synthesis of RNA. They come in a variety of sizes, including the large “hammerhead”. They range in size from the large?hammerhead? to the smaller?minizymes’. These are synthetic constructs that contain the minimum structures necessary for activity. Similar properties were also found in DNA-based enzymes (deoxyribozymes or DNAzymes). They have the potential to be used as therapeutic agents due to their ability to cut and recognize specific mRNA molecules. A ribozyme that catalyzes the cleavage a specific mRNA could be used as a therapeutic agent in much the same way as an antisense nucleic acids. However, a single ribozyme can destroy many copies. A synthetic ribozyme (Angiozyme?) It cleaves mRNA encoding a subtype of VEGF receptor. This is currently being studied in clinical trials to treat cancer.
“RNA interference (RNAi), is the phenomenon of gene specific post-transcriptional silencing using double-stranded RNA oligomers. Nature 2001, 411: 494-498; Caplen et al., Proc. Natl. Acad. Sci. U.S.A. 2001, 98: 9742-9747). Antisense oligonucleic acid and ribozymes have the potential to be therapeutic agents, reducing harmful protein expression by small inhibitoryRNAs (siRNAs). A protein complex, the RNA induced silencing compound, recognizes siRNA as double-stranded. This complex strips away one strand, facilitates hybridization with the target target mRNA and then cleaves that strand. For the same reason DNA-based vectors are capable of creating siRNA within cells. Short hairpin RNAs are also useful for this purpose. SiRNAs that can target specific genes, both endogenously or exogenously, have been described. See Paddison et al. Proc. Natl. Acad. Sci. U.S.A., 2002, 99: 1443-1448; Paddison et al., Genes & Dev. 2002, 16: 948-958; Sui et al. Proc. Natl. Acad. Sci. U.S.A. 2002. 8: 5515-5520. Brummelkamp and co., Science 2002. 296: 553-553.
“The term “nucleic acid-based therapeutic agents?” Three classes of compounds are included in the definition. This term includes all pharmaceutically acceptable salts and esters, prodrugs and codrugs as well as protected forms of the compounds, analogues and derivatives. The first class is collectively referred to as “antisense nucleic acid”. The first class, also known as?antisense nucleic acid, is composed of nucleic acids with 50 or less monomers that can be hybridized in a sequence-specific fashion to a single-stranded or targeted DNA molecule. This class includes ordinary DNA, RNA oligomers and DNA with modified backbones. It also includes phosphorothioates and methylphosphonates as well as peptide nucleic Acids, 2?-deoxy derivatives and nucleic Acid Oligomers that have chemically modified purine bases and pyrimidine bases. These oligomers can be lipophilically modified and/or pEGylated to alter their pharmacodynamics. This class also includes Oligomers that act as precursors to such agents, such hairpin RNAs, which are converted into siRNAs in cells.
Aptamers are the second type of nucleic acids-based therapeutic agents. Aptamers are nucleic acid oligomers with 50 or less monomer units that can bind with structural specificity to non-oligonucleotide target molecules or an oligonucleotide using sequence-specific hybridization. This class includes DNA and RNA Aptamers. Modifications of these nucleic acids include mirror-image DNA (?Spiegelmers) and RNA (?) ), peptide nucleic and nucleic acid Oligomers that have been chemically modified in any other way than what is described above. Any of these species can also contain chemically modified purines or pyrimidines, and may also be lipophilically modified (or PEGylated). M. Rimmele’s Chembiochem. 2003, 4: 963-71, and A. Vater, Curr. Opin. Drug Discov. Devel. 2003, 6: 253-61 to see recent reviews of aptamer tech. You will appreciate that members of the second class have, in addition to a structure-specific affinity for target molecules, a sequence-specific affinity towards a putative DNA sequence or RNA sequence.
“The third category of nucleic-acid-based therapeutic agents, also known as ‘nucleic acids enzymes?, is the third. This class includes nucleic acid-based therapeutic agents that can recognize and catalyze the cleavage target RNA molecules in a sequence-specific way. This class also includes hammerhead and minimized hammerheads (or minizymes). ), ?10-23? deoxyribozymes (?DNAzymes? ), and other similar. The class also includes chemically modified catalytic species, just like antisense molecules and aptamer compounds.
“Pharmaceutically acceptable salts” is a term that refers to the compounds of the invention. The term “pharmaceutically acceptable salts” refers to the physiologically and pharmaceutically acceptable salts for the compounds of the invention.
“A ?protein coding sequence? A sequence that?encodes? A sequence that?encodes? a specific polypeptide. A start codon at the 5 determines the boundaries of the coding sequence. (amino terminus) and a translation end codon at 3? (carboxyl) terminus. A coding sequence may include, but not be limited to, cDNA derived from prokaryotic and eukaryotic genomic mRNA, as well as cDNA sequences derived from prokaryotic and eukaryotic DNA. It can also include synthetic DNA sequences. Usually, a transcription termination sequence is located at 3? To the coding sequence.”
“The term ‘RNAi construct’ is used herein. It is a generic term that includes siRNA, hairpinRNA and other RNA species that can be cleaved live to make siRNAs. RNAi constructs also include expression vectors, also known as RNAi expression Vectors. These vectors can give rise to transcripts that form dsRNAs or RNAi in cells and/or transcripts that can be converted to siRNAs.
“As used in this document, the term “vector”? A nucleic acids molecule that can transport another nucleic acids to which it is linked. A genomic integrated vector (or?integrated vector) is one type of vector. These can be integrated into the host cell’s chromosomal genome. An episomal vector is another type of vector. It is a nucleic acids capable of extra-chromosomal reproduction. These vectors are known as expression vectors. In this specification, “plasmid” is defined as: If the context is not clear,?plasmid? und?vector are interchangeable. Unless otherwise stated, they are interchangeable. Expression vectors can contain regulatory elements that control transcription. These elements may be derived from insect, mammalian, or microbial genes. A host’s ability to reproduce is usually conferred by an origin. Additionally, a selection gene that facilitates recognition of transformants can be included. You can use vectors derived from viruses such as retroviruses and adenoviruses.
“The present disclosure is about the use of antisense nuclear acid to reduce the expression of a targeted disease-related proteins. An antisense nucleic acids can be delivered to cells, such as as expression plasmids that produce RNA that is complementary to the cellular portion of the mRNA that encodes the targeted disease-related proteins. The construct can also be an oligonucleotide, which is ex vivo generated and introduced into cells to inhibit expression. This is done by combining the mRNA or genomic sequences that encode the targeted disease-related proteins with it. These oligonucleotides can be optionally modified to resist endogenous exonucleases or endonucleases. Exemplary nucleic acids molecules that can be used as antisense Oligonucleotides include phosphoramidate and phosphothioate. (See U.S. Pat. Nos. 5,176,996 and 5,264,564, respectively. Van der Krol and colleagues (1988), Biotechniques 6; 958-976, and Stein et. al. (1988), Cancer Res 48: 2659?2668, have reviewed the general approaches for constructing oligomers that are useful in nucleic acids therapy.
“In other embodiments, the application relates to the use RNA interference (RNAi), to effect knockdown the targeted gene. RNAi constructs are double-stranded RNA which can be used to block the expression of a targeted gene. RNAi constructs may contain RNA that is identical or substantially the same as the target nucleic acids sequence or RNA that is identical or substantially the same as a specific region of the target sequence.
“Optionally, RNAi constructs can contain a nucleotide sequencing that hybridizes under physiologic circumstances of the cell with the nucleotide structure of at least a part of the mRNA transcript (the?target?) gene). Double-strandedRNA must be identical to natural RNA in order to be able to induce RNAi. The invention allows for embodiments that can tolerate sequence variations due to genetic mutations, polymorphic sites or evolutionary divergence. Although the number of nucleotide mismatches that can be tolerated between the target sequence sequence and the RNAi construct sequencing may reach 1 in 5, it is preferably less than 1 in 10. The most important mismatches in siRNA duplex’s center are the most serious and can essentially stop cleavage. Nucleotides located at the 3? Nucleotides at the 3? end of siRNA strands that are complementary to target RNA don’t significantly affect target recognition specificity. Sequence comparison and alignment algorithms are available to optimize sequence identity (see Gribskov & Devereux, Sequence Analysis Primer Stockton Press 1991 and references therein). The Smith-Waterman algorithm, which is implemented in the BESTFIT software program with default parameters (e.g. University of Wisconsin Genetic Computing Group), can be used to calculate the percent difference between nucleotide sequences. Preferably, between 90% and 100% sequence identity is maintained between the inhibitoryRNA and the target gene portion. Alternativly, the functional definition of the duplex region in RNA could be that it is a nucleotide sequence capable of hybridizing with target gene transcripts after hybridization at 50.degree for 12-16 hours. C. to 70.degree. C. in 400mM NaCl and 40mM pH 6.4 PIPES pH 6.4. Followed by washing with 1.0 mM EDA.
The double-stranded structure can be made by one self-complementary or two complementary RNAs strands. The dsRNA can be formed inside or outside the cell. You can introduce the RNA in a quantity that allows for delivery of at most one copy per cell. Double-stranded material can be administered in higher doses (e.g. at least 5, 10, 100 or 500 copies per cell), which may result in more effective inhibition. However, lower doses might prove useful for certain applications.
“The subject RNAi constructs may be?small-interfering RNAs?” Or?siRNAs. These nucleic acid are shorter than 50 and usually have a length of 19-30 nucleotides, but more preferably 21-23. SiRNAs recruit nuclease clusters and guide complexes to target mRNA by matching to specific sequences. The siRNAs in the protein complex degrade the target mRNA. A particular embodiment of siRNA molecules 21-23 includes a 3? The hydroxyl group. The siRNA constructs may be made by processing longer double-stranded RNAs in certain embodiments. One embodiment uses the Drosophila In vitro System. This embodiment combines dsRNA with a soluble extract derived directly from Drosophila embryos, thereby creating a combination. This combination is kept under conditions where the dsRNA can be processed to RNA molecules ranging from 21 to 23 nucleotides. There are many techniques that can be used to purify siRNA molecules, including gel electrophoresis. Non-denaturing methods such as column chromatography and size exclusion chromatography can also be used to purify siRNAs.
The chemical synthesizer or recombinant nucleic acids techniques can both be used to produce RNAi constructs. The cell treated may have an endogenous RNApolymerase that mediates transcription in vivo. Cloned RNApolymerase can also be used to produce transcription in vitro. Modifications to the phosphate sugar backbone or nucleoside may be made to RNAi constructs. These modifications can reduce susceptibility to cellular nuclear nucleases, increase bioavailability, improve formulation characteristics and/or alter other pharmacokinetic properties. The phosphodiester linkages in natural RNA can be modified to include at most one nitrogen or sulfur heteroatom.
Modifications to RNA structure can be tailored to permit specific genetic inhibition, while avoiding a broad response to dsRNA. Bases can also be modified to inhibit the activity of adenosine desminase. You can make the RNAi construct either enzymatically, or through partial/total organic synthesis. Any modified ribonucleotide may also be created by organic synthesis or in vitro enzymatic synthesis. You can modify RNAi constructs by chemically modifying RNA molecules (see Heidenreich et. al. (1997) Nucleic Acids Res. 25: 776-780; Wilson et al. (1994) J. Mol. Recog. 7: 89-98; Chen et al. (1995) Nucleic Acids Res. 23: 2661-2668; Hirschbein et al. (1997) Antisense Nucleic Acid Drug, Dev. 7: 55-61). For example, the backbone of an RNAi construct can be modified with phosphorothioates, phosphoramidate, phosphodithioates, chimeric methylphosphonate-phosphodiesters, peptide nucleic acids, 5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g., 2?-substituted or 2?-deoxy ribonucleosides, .alpha.-configurations, etc.).”
“In certain embodiments, at most one strand of siRNA molecules could have a 3? Overhangs can be as short as 1 to 6 nucleotides. The preferred number of overhangs is 3. overhangs are 1-3 nucleotides in length. One strand may have a 3? The overhang is different for each strand. One strand may be blunt-ended, while the other may have an overhang. Each strand may have different overhang lengths. The 3? can be used to enhance siRNA stability. Overhangs can be protected from degradation. One embodiment of the invention includes purine nucleotides such as adenosine and guanosine nucleotides to stabilize the RNA. Alternately, you can substitute pyrimidine nucleotides with modified analogues (e.g. substitution of uridine ucleotide 3). Overhangs caused by 2?-deoxythymidine may be tolerated, but this will not affect the effectiveness of the RNAi. In the absence of a 2? The absence of a 2?hydroxyl increases the nuclease resistance to the overhang in tissue culture medium and may also be beneficial in vivo.
“The RNAi construct may also come in the form a long, double-stranded RNA that is intracellularly digested to create a siRNA sequence. The RNAi construct could also be a hairpin RNA. The art of siRNA production is possible by processing hairpinRNAs in cells. Exogenous hairpin RNAs are possible to be synthesized or formed in vivo by transcribing RNA polymerase II promoters. For example, Paddison et. al. Genes Dev 2002, 16: 948-58, McCaffrey et. Nature 2002, 418, 38-9, McManus et., RNA 2002, 8: 842-50, Yu et. al. Proc. Natl. Acad. Sci. USA, 2002, 99: 6047-52). These hairpin RNAs can be engineered in cells, or in animals to ensure the continuous and stable suppression a desired gene.
“PCT Application WO 01/77350 describes an example vector for bidirectional transcription of a transcript to produce both sense and antisense transcripts from the same transgene within a eukaryotic cells. In certain embodiments, this invention provides a recombinant virus with the following characteristics: It comprises a viral replicon that has two overlapping transcription unit arranged in opposing orientations and flanking a transgene to produce an RNAi construct. The two overlapping transcription units are able to generate both sense and antisense transcripts from the same transgene in a host cells.
“In another embodiment, this disclosure refers to the use ribozyme compounds designed to catalytically clear an mRNA transcript to stop translation (see, for example, PCT International Publication W90/11364, Oct. 4, 1990; Sarver and al., 1990 Science 247: 1222-21225; and U.S. Patent. No. 5,093,246). Any ribozyme capable of cleaving the target mRNA at a site specific recognition sequence can be used, but hammerheads ribozymes are preferred. Hammerhead ribozymes cleave target mRNAs at specific locations that are dictated by flanking areas that form complementary base pair with the target mRNA. The only requirement is that the target’s mRNA contain the following sequence: 5?-UG-3?. It is well-known that hammerheads ribozymes can be constructed and produced. This is discussed in Haseloff & Gerlach 1988, Nature 334: 585-591. The ribozymes described in the present invention also include RNA Endoribonucleases (Cech-type ribozymes). The present invention also includes RNA endoribonucleases (?Cech-type ribozymes?) WO88/04300 from University Patents Inc. (Been and Cech 1986, Cell 47: 207-216)
“A further embodiment of the invention is the use DNA enzymes to inhibit the expression of a targeted genes. DNA enzymes combine some of the mechanistic characteristics of both antisense as ribozyme technology. DNA enzymes are designed to recognize a specific target sequence of nucleic acids, much like antisense oligonucleotides. However, they are catalytic and cleave that target nucleic acids much like ribozymes. In short, in order to create a DNA enzyme that recognizes and cleaves the target nucleic acids, someone skilled in the art must first identify the target sequence. The sequence should be a G/C rich, approximately 18-22 nucleotides. A high G/C content will ensure a stronger interaction between DNA enzyme and target sequence. The specific antisense sequence that will target DNA enzyme to the message is split so that it includes the two arms of DNA enzyme. The DNA enzyme loop is then placed between these two specific arms. U.S. Pat. describes methods for making and administering DNA enzymes. No. 6,110,462.”
The methods described herein can be used to deliver a variety molecules including, but not limited, small molecules (including those that are not optimally cell-permeability), lipids and nucleotides as well as nucleic acids, nucleotides polynucleotides polynucleotides polynucleotides polynucleotides polynucleiotides, nucleic acid, polynucleotides polynucleotides to polyamines, hormones, or polyamines to cross cellular membranes. Non-limiting examples of polynucleotides that can be delivered across cellular membranes using the compounds and methods of the invention include short interfering nucleic acid (siNA), antisense, enzymatic nucleic acid molecules, 2?,5?-oligoadenylate, triplex forming oligonucleotides, aptamers, and decoys. You can deliver biologically active molecules such as antibodies (e.g. monoclonal or chimeric, humanized, etc. ), cholesterol and hormones, antivirals and peptides.
The compounds, compositions and methods of invention can increase the delivery or availability biologically active molecules to cells and tissues (e.g. siNAs and siRNAs and siRNA inhibitors), compared with delivery of the molecules to cells or tissues in the absence of the compositions and methods. The presence of compounds and compositions from the invention can increase the biologically active molecules in cells, tissues, and organisms compared to when they are absent.
“The term ‘ligand? refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter that is capable of interfacing with another compound. “The term?ligand” refers to any compound or molecular, such a drug or peptide, hormone or neurotransmitter, that can interact with another compound such as a receptor either directly or indirectly. A receptor that interacts directly or indirectly with a ligand may be found on the cell’s surface. Alternately, it can also be an intracellular receptor. Interaction between the receptor and the ligand can lead to a biochemical response or a physical interaction. Galactose, galactosamine and N-acetylgalactosamine are all examples of ligands. A linker molecule can attach the ligand to a compound according to the invention. Examples include an amido, carbonyl; ester, protein, disulphide or silane, nucleosides, nucleosides, nucleosides, and nucleosides. One embodiment of the linker is a biodegradable one.
“Linkers”
A variety of linkers can be used to connect the substrate that is capable of being transported to active agent. You can use both degradable or cleavable links.
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