[go: up one dir, main page]

WO2013082515A2 - Aptamères d'acides nucléiques dirigés sur des récepteurs de surface et procédés d'utilisation - Google Patents

Aptamères d'acides nucléiques dirigés sur des récepteurs de surface et procédés d'utilisation Download PDF

Info

Publication number
WO2013082515A2
WO2013082515A2 PCT/US2012/067423 US2012067423W WO2013082515A2 WO 2013082515 A2 WO2013082515 A2 WO 2013082515A2 US 2012067423 W US2012067423 W US 2012067423W WO 2013082515 A2 WO2013082515 A2 WO 2013082515A2
Authority
WO
WIPO (PCT)
Prior art keywords
aptamer
trkb
nucleic acid
agonist
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2012/067423
Other languages
English (en)
Other versions
WO2013082515A3 (fr
Inventor
James O. Mcnamara
Yangzhong Huang
Bin Gu
James O. MCNAMARAII
Paloma H. Giangrande
Frank Jeyson HERNANDEZ
Katie R. FLENKER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Iowa Research Foundation UIRF
Duke University
Original Assignee
University of Iowa Research Foundation UIRF
Duke University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Iowa Research Foundation UIRF, Duke University filed Critical University of Iowa Research Foundation UIRF
Publication of WO2013082515A2 publication Critical patent/WO2013082515A2/fr
Publication of WO2013082515A3 publication Critical patent/WO2013082515A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Definitions

  • binding affinity of the aptamers herein with respect to targets and other molecules is defined in terms of 3 ⁇ 4 ⁇ ⁇
  • the value of this dissociation constant can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci, M., et al., Byte (1984) 9:340-362.
  • binding activity and “binding affinity” are meant to refer to the tendency of a ligand molecule to bind or not to bind to a target.
  • modified purines are known to include, but are not limited to, 2'-0-methyl nucleotides; and modified pyrimidines are known to include, but are not limited to, 2'-deoxy-2'- fluoro nucleotides or 2'-deoxy-2'-fluoroarabino nucleotides.
  • the aptamers of the invention are specifically binding oligonucleotides, wherein "oligonucleotide” is as defined herein.
  • oligonucleotides include not only those with conventional bases, sugar residues and internucleotide linkages, but also those that contain modifications of any or all of these three moieties.
  • a consensus sequence can be. as short as three nucleotides long. It also can be made up of one or more noncontiguous sequences with nucleotide sequences or polymers of hundreds of bases long interspersed between the consensus sequences. Consensus sequences can be identified by sequence comparisons between individual aptamer species, which comparisons can be aided by computer programs and other tools for modeling secondary and tertiary structure from sequence information. Generally, the consensus sequence will contain at least about 3 to 20 nucleotides, more commonly from 6 to 10 nucleotides.
  • Consensus sequence means that certain positions, not necessarily contiguous, of an oligonucleotide are specified. By specified it is meant that the composition of the position is other than completely random. Not all oligonucleotides in a mixture can have the same nucleotide at such position; for example, the consensus sequence can contain a known ratio of particular nucleotides.
  • neuropsychiatric disorder refers to a pathological condition relating to the brain and/or nervous system.
  • the disorder can be regulated by the TrkB signaling pathway.
  • Such conditions include, but are not limited to, stroke, anxiety, epilepsy, head trauma, migraine, obesity, chronic neuropathic pain, acute neuropathic pain, schizophrenia, diseases such as Alzheimer's and Huntington's disease, depression and addiction.
  • the terms "neurological or neuiOpsychiatric disease” and “neurological or neuropsychiatric disorder” can be used interchangeably.
  • epilepsy syndromes by location or distribution of seizures (as revealed by the appearance of the seizures and by EEG) and by cause. Syndromes are divided into localization-related epilepsies, generalized epilepsies, or epilepsies of unknown localization.
  • the term "pain” refers to the basic bodily sensation induced by a noxious stimulus, received by naked nerve endings, characterized by physical discomfort (e.g., pricking, throbbing, aching etc.) and typically leading to an evasive action by the individual.
  • the term “chronic neuropathic pain” refers to a complex, chronic pain state that is usually accompanied by tissue injury wherein the nerve fibers themselves may be damaged, dysfunctional or injured. These damaged nerve fibers send incorrect signals to other pain centers.
  • the impact of nerve fiber injury includes a change in nerve function both at the site of injury and areas around the injury.
  • neurotrophin and “neurotrophic factor” and their grammatical variants are used interchangeably, and refer to a family of polypeptides comprismg nerve growth factor (NGF) and sequentially related homologs.
  • NGF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • NT-4/5 neurotrophins-4 and -5
  • the linker comprises a length of about 3 to about 20 nanometers (nm), and more preferably, from about 5 to about 10 nm.
  • linkers may include, but are not limited to, carbon chains having a length of from about 10 carbons to about 20 carbons, nucleic acid molecules comprised of between 10 to 40 nucleic acid bases (single-stranded) or base pairs (double-stranded), or a combination thereof.
  • Multimer is used herein, for purposes of the specification and claims, to mean two or more aptamers that are linked together.
  • a multimer may comprise a dimer, trimer, tetramer, etc.
  • the aptamer molecules in a multimer may be of the same nucleic acid sequence and/or binding specificity, as compared to other aptamer molecules present as part of the multimer.
  • the aptamer molecules in a multimer may be of a different nucleic acid sequence and/or binding specificity as compared to other aptamer molecules present as part of the multimer.
  • Mulitmers may be connected to each other by one or more linkers.
  • Substantially homologous also includes base pair flips in those areas of the nucleic acid aptamers that include base pairing regions.
  • Substantially the same ability to bind BDNF receptors e.g., TrlcB
  • the affinity is within two orders of magnitude of the affinity of the nucleic acid aptamers described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence is substantially homologous to and has substantially the same ability to bind BDNF receptors (e.g., TrkB), as the sequences identified herein.
  • a nucleic acid aptamer of the present invention selectively binds a BDNF receptor (e.g., TrkB), thereby exhibiting potent BDNF receptor (e.g., TrkB) partial agonistic activity and neuroprotective effects in cultured cortical neurons.
  • BDNF receptor e.g., TrkB
  • this aptamer comprises the sequence, 5 ' -GGGAGGACGAUGCGGUCGUAUUAUCCGCUGCA CGCCAGACGACUCGCCCGA-3' (SEQ ID NO:l):
  • all cytidines are 2'-deoxy-2' fluoro cytidine and all uridines are 2'-deoxy-2'-fluorouridine.
  • the dissociation constant ranges from about 100 pM to about 10 nM.
  • the dissociation constant ranges from about 400 pM to about 10 nM, and can optionally comprise any value within the range, e.g. about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1 nM, about 2.5 nM, or about 5 nM.
  • oligonucleotides that contain that sequence can be made by conventional synthetic or recombinant techniques. These aptamers can also function as ligand-specific aptamers of this invention. Such an aptamer can contain the entire nucleotide sequence of an isolated aptamer, or can contain one or more additions, deletions or substitutions in the nucleotide sequence, as long as a consensus sequence is conserved. A mixture of such aptamers can also function as ligand-specific aptamers, wherein the mixture is a set of aptamers with a portion or portions of their nucleotide sequence being random or varying, and a conserved region that contains the consensus sequence. Additionally, secondary aptamers can be synthesized using one or more of the modified bases, sugars and linkages described herein using conventional techniques and those described herein.
  • aptamers can be sequenced or mutagenized to identify consensus regions or domains that are participating in aptamer binding to ligand, and/or aptamer structure. This information is used for generating second and subsequent pools of aptamers of partially known or predetermined sequence. Sequencing used alone or in combination with the retention and selection processes of this invention, can be used to generate less diverse oligonucleotide pools from which aptamers can be made. Further selection according to these methods can be carried out to generate aptamers having preferred characteristics for diagnostic or therapeutic applications. That is, domains that facilitate, for example, drug delivery could be engineered into the aptamers selected according to this invention.
  • this invention is directed to making aptamers using screening from pools of non-predetermined sequences of oligonucleotides, it also can be used to make second- generation aptamers from pools of known or partially known sequences of oligonucleotides.
  • a pool is considered diverse even if one or both ends of the oligonucleotides comprising it are not identical from one oligonucleotide pool member to another, or if one or both ends of the oligonucleotides comprising the pool are identical with non-identical intermediate regions from one pool member to another.
  • Structural features can be considered in generating a second (less random) pool of oligonucleotides for generating second round aptamers.
  • Different regions of a polypeptide interact with each other through hydrophobic and electrostatic interactions and also by formation of salt bridges, disulfide bridges, etc. to form the secondary and tertiary structures.
  • Defined conformations can be formed within the protein organization, including beta sheets, beta barrels, and clusters of alpha helices.
  • Optimal binding sequences will be those which exhibit high relative affinity for the ligand, i.e., affinity measured in 3 ⁇ 4 in at least in the nanomolar range, and, for certain drug applications, the nanomolar or picomolar range.
  • studying the binding energies of aptamers using standard methods known generally in the art can be useful generally, consensus regions can be identified by comparing the conservation of nucleotides for appreciable enhancement in binding.
  • an aptamer Once an aptamer has been identified, it can be used, either by linkage to, or use in combination with, other aptamers identified according to these methods. One or more aptamers can be used in this manner to bind to one or more targets.
  • a method of modulating the biological activity of a BDNF receptor (e.g., Trl B) in a subject comprises: (a) administering to a subject an effective amount of a nucleic acid aptamer that selectively binds a surface receptor (e.g., TrkB).
  • the nucleic acid aptamer has a dissociation constant for the molecule of about 20 nM or less; and (b) modulating the biological activity of the BDNF receptor in the subject through the administering of the nucleic acid aptamer in step (a).
  • a method of treating neurological or neuropsychiatric disorder comprises administering an effective amount of a nucleic acid aptamer that selectively binds a BDNF receptor (e.g., TrkB), whereby neurological or neuropsychiatric disorder in the subject is treated.
  • a BDNF receptor e.g., TrkB
  • the nucleic acid aptamer has a dissociation constant for the BDNF receptor of about 20 nM or less.
  • neurological or neuropsychiatric disorder is selected from the group consisting of stroke, anxiety, epilepsy, head trauma, migraine, obesity, chronic neuropathic pain, acute neuropathic pain, schizophrenia, Alzheimer's disease, Huntington's disease, depression and addiction.
  • neurological or neuropsychiatric disorder is Huntington's disease.
  • the patient treated in the present invention in its many embodiments is desirably a human subject, although it is to be understood that the principles of the invention indicate that the invention is effective with respect to all vertebrate species, including warm-blood vertebrates (e.g. birds and mammals), which are intended to be included in the term "subject".
  • warm-blood vertebrates e.g. birds and mammals
  • the terms "subject” and “patient” are used interchangeably.
  • a mammal is understood to include any mammalian species in which treatment of neurological or neuropsychiatric disease is desirable, particularly agricultural and domestic mammalian species.
  • Contemplated is the treatment of mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economical importance (animals raised on farms for consumption by humans and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans such as cats and dogs), swine (pigs, hogs, and wild board), nuninants (such as cattle, oxen, sheep, giraffes, deer goats, bison, and camels), and horses.
  • fowl i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical importance to humans.
  • the present method for treating neurological or neuropsychiatric disorder in a tissue contemplates contacting a tissue in which neurological or neuropsychiatric disorder (e.g., pain) is occurring, or is at risk for occurring, with a composition comprising a therapeutically effective amount of a nucleic acid aptamer capable of binding a BDNF receptor (e.g., Trl B).
  • a composition comprising a therapeutically effective amount of a nucleic acid aptamer capable of binding a BDNF receptor (e.g., Trl B).
  • the method comprises administering to a patient a therapeutically effective amount of a physiologically tolerable composition containing the RNA aptamer.
  • neurological or neuropsychiatric disorder is selected from the group consisting of stroke, anxiety, epilepsy, head trauma, migraine, obesity, chronic neuropathic pain, acute neuropathic pain, schizophrenia, Alzheimer's disease, Huntington's disease, depression and addiction. In other embodiment, neurological or neuropsychiatric disorder is Huntington's disease.
  • the dosage ranges for the administration of the nucleic acid aptamer depend upon the form of the modulator, and its potency, as described further herein, and are amounts large enough to produce the desired effect in which a BDNF receptor, e.g., TrkB, is modulated, which can correspondingly ameliorate neurological or neuropsychiatric disorder(s) and the symptoms of neurological or neuropsychiatric disorder(s).
  • the dosage should not be so large as to cause adverse side effects.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be deteraiined by one of skill in the art. The individual physician in the event of any complication can also adjust the dosage.
  • a therapeutically effective amount is an amount of an RNA aptamer sufficient to produce a measurable modulation of the BDNF signaling molecule, e.g., TrkB, in the tissue being treated. Modulation of molecules such as TrkB can be measured in situ by immunohistochemistry by methods disclosed in the Laboratory Examples, or by other methods known to one skilled in the art. By the term “modulate” and grammatical variations thereof, it is intended an increase, decrease, or other alteration of any or all biological activities or properties a target.
  • TrkB modulator can take the form of RNA aptamers, it is to be appreciated that the potency, and therefore an expression of a "therapeutically effective" amount can vary. However, as shown by the methods presented in the Laboratory Examples, one skilled in the art can readily assess the potency of a candidate RNA aptamer of this invention.
  • RNA aptamers of the present invention can be administered parenterally by , injection or by gradual infusion over time.
  • tissue to be treated can typically be accessed in the body by systemic administration and therefore most often treated by intravenous administration of therapeutic compositions, other tissues and delivery techniques are provided where there is a likelihood that the tissue targeted contains the target molecule.
  • an RNA aptamer of the present invention can be administered orally, nasally (e.g., via a nebulizer), intravenously, intraperitoneally, intramuscularly, subcutaneously, intra-cavity, transdermally, and other techniques.
  • compositions of the present invention contemplates therapeutic compositions useful for practicing the therapeutic methods described herein.
  • Therapeutic compositions of the present invention contain a physiologically tolerable carrier together with a nucleic acid aptamer as described herein, dissolved or dispersed therein as an active ingredient.
  • the therapeutic composition is not immunogenic when administered to a subject for therapeutic purposes.
  • the term "pharmaceutically acceptable carrier” includes any compound or composition or carrier medium useful in any one or more of administration, delivery, storage, or stability of a nucleic acid aptamer described herein.
  • These carriers are known in the art to include, but are not limited to, water, saline, suitable vehicle (e.g., liposome, microparticle, nanoparticle, emulsion, capsule and the like), buffer, medical parenteral vehicle, excipient, aqueous solution, suspension, solvent, emulsions, detergent, chelating agent, solubilizing agent, diluent, salt, colorant, polymer, hydrogel, surfactant, emulsifier, adjuvant, filler, preservative, stabilizer, oil, and the like as broadly known in the pharmaceutical art.
  • compositions comprising a nucleic acid aptamer of the present invention can be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation can be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the aptamer. Such compositions can contain admixtures of more than one aptamer.
  • the aptamers herein described in detail can form the active ingredient, and may be topically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrup, suppositories, gels and the like, and consistent with conventional pharmaceutical practices.
  • carrier suitable pharmaceutical diluents, excipients or carriers
  • suitable pharmaceutical diluents, excipients or carriers suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrup, suppositories, gels and the like, and consistent with conventional pharmaceutical practices.
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders include without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
  • the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
  • suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
  • Other dispersing agents include glycerin and the like.
  • sterile suspensions and solutions are desired.
  • Isotonic preparations that generally contain suitable preservatives are employed when intravenous adrrrinistration is desired.
  • Topical preparations containing the active drag component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, sldn lotions, and shampoos in cream or gel formulations.
  • carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, sldn lotions, and shampoos in cream or gel formulations.
  • the compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
  • the compounds of the present invention can also be coupled with soluble polymers as targetable drug carriers.
  • soluble polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl- eneoxidepolylysine substituted with palmitoyl residues.
  • the compounds of the present invention can be coupled (preferably via a covalent linkage) to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polyethylene glycol (PEG), polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • PEG polyethylene glycol
  • polylactic acid polyepsilon caprolactone
  • polyhydroxy butyric acid polyorthoesters
  • polyacetals polydihydro-pyrans
  • polycyanoacrylates polycyanoacrylates
  • cross-linked or amphipathic block copolymers of hydrogels for example, polyethylene glycol (PEG), polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans
  • a method of identifying a nucleic acid aptamer to a receptor e.g., a receptor tyrosine kinase
  • a receptor e.g., a receptor tyrosine kinase
  • Products, i.e. aptamers, produced or identified by a method of the present invention are also provided.
  • the present invention provides a method of identifying a nucleic acid aptamer having agonistic (or partial agonistic) activity comprising (a) contacting a nucleic acid aptamer with cells comprising a receptor to which the nucleic acid aptamer binds; and (b) detecting a cell activity, which activity is initiated by cell signaling resulting from the binding of the nucleic acid aptamer to the ligand; wherein detection of the cell activity is an indication that the nucleic acid aptamer comprises an agonist (or partial agonist) aptamer.
  • the present invention provides for a method of producing a nucleic acid aptamer having agonist (or partial agonist) activity comprising: (a) contacting a nucleic acid aptamer with cells comprising a receptor to which the nucleic acid aptamer binds; wherein the receptor comprises a receptor tyrosine kinase; and where crosslinking of the receptor by the nucleic acid aptamer results in cell signaling; (b) detecting a cell activity, which activity is initiated directly or indirectly by cell signaling resulting from the binding of the nucleic acid aptamer to the receptor; wherein detection of the cell activity is an indication that the nucleic acid aptamer comprises an agonist (or partial agonist) aptamer; and (c) synthesizing the agonist (or partial agonist) aptamer, in producing the agonist (or partial agonist) aptamer.
  • the receptor is a tyrosine kinase receptor.
  • the receptor is a BNDF receptor (e.g., TrkB).
  • the aptamer mixture can comprise a candidate mixture of any aptamer as defined herein, including but not limited to nucleic acids.
  • the candidate mixture of nucleic acids comprises single strand nucleic acids.
  • the single stranded nucleic acids can comprise deoxyribonucleic acids.
  • EXAMPLE 1 R A Aptamers and Surface Receptors.
  • BDNF was purchased from Millipore (formerly Chemicon; Billerica, MA). Other reagents were obtained from Sigma (St. Louis, MO) unless specified otherwise.
  • TrkB mammalian plasmid pcDNA3-FLAG-TrgB has been described previously (see, e.g., Huang, E.J. and Reichardt, L., supra).
  • Cortical neuron/glia mixed cultures were prepared from embryonic 18 (El 8) pups of pregnant Sprague-Dawley rat (Charles River; Durham, NC) as described previously (see, e.g., Huang, E.J. and Reichardt, L., supra). The neurons were cultured in Neurobasal with B27 supplement and Glutamax (Invitrogen; Carlsbad, CA) for 12- 24 days in vitro (DIV). HEK293 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum.
  • HEK293 cells were plated 12 h before transfection at a density of 5x10 5 cells/well of 6-well culture plate and were transfected using Lipofectamine method (GIBCO BRL; Carlsbad, CA). 24 h after transfection, G418 (lmg/ml) was added to medium for 14 days. A single cell clone was selected and expanded. Ariimals were handled according to National Institutes of Health Guide for the Care and Use of the Laboratory Arrimals and approved by Duke University Animal Welfare Committee.
  • a template DNA oligo (5 ' -TCGGGCGAGTCGTCTGNNNNNNNNN NNNNNNNNCCGCATCGTCCTCCC-3') (SEQ ID NO:2) and a 5'-oligo (5'-TAATAC GACTCACTATAGGGAGGACGATGCGG) (SEQ ID NO:3) (both synthesized by Integrated DNA Technologies (IDT), Coralville, IA) were annealed and extended with Taq Polymerase (Denville Scientific, Inc.; Metuchen, NJ).
  • ImM ATP Gibcoadenophosphate
  • GTP Gibco-GTP
  • 3 mM 2'fluoro-CTP Trilink; San Diego, CA
  • 3 mM 2'-fluor-UTP Trilink; San Diego, CA
  • RNA recovered from cells after each cell- internalization procedure was reverse transcribed with a 3'-oligo (5'-TCGGG CGAGTCGTCT-3 ') (SEQ ID NO:4) (IDT; Coralville, IA) using AMV Reverse Transcriptase (Roche; San Francisco, CA); the RT reaction was then used as template in a 1 ml, 20 cycle PCR with 5'-oligo and 3'-oligo to generate the transcription template for the subsequent round of selection.
  • RNA 750 picomoles of library RNA were used for the first round; 500 picomoles of RNA were used in each subsequent round.
  • the procedure for each round was as follows: A 150mm dish of HEK cells (-90% confluence) was first blocked by washing the cells twice with Dulbecco's Phosphate-Buffered Saline (DPBS) containing calcium and magnesium (Gibco; San Francisco, CA) followed by incubation at 37°C for 15 minutes in 15mls DPBS supplemented with 100 ⁇ g/ml yeast tRNA (Invitrogen; San Francisco, CA).
  • DPBS Dulbecco's Phosphate-Buffered Saline
  • the DPBS/tRNA was replaced with 15 mis of DPBS containing the library and 100 ⁇ g/ml yeast tRNA. After 15 minutes incubation at 37°C, the supernatant was transferred to a centrifuge tube and spun at 2,500rpms in a table-top centrifuge in order to pellet cellular debris. Following centrifugation, the supernatant was transferred to a 150mm dish of TrkB-expressing HEK cells (-90% confluent) that had been blocked with tRNA as described for the HEK cells above. After a 20-min incubation at 37°C, with periodic gentle mixing, the supernatant was discarded.
  • RNAse A Fermentas; Gen Burnie, MD
  • RNA was then purified with phenol/chloroform/isoamyl alcohol and chloroform extractions, followed by ethanol precipitation. Each dried RNA pellet was dissolved in 50 ⁇ water and stored at -20°C. A similar "pre-clearing" strategy was implemented for each subsequent round of selection.
  • Primer A.8 SEQ ID NO: 5
  • the PCR product was purified with a Qiagin Miniprep column and then combined with PCR products generated in the same manner (but with distinct bar codes) and submitted to the U. of Minnesota sequencing facility.
  • C4-3 TrkB Aptamer Affinity The immobilization of C4-3 aptamer was performed by the streptavidin-biotin coupling method.
  • C4-3 aptamer was previously biotinylated at 3 '-end by periodate oxidation as previously described (Qin, P.Z. and Pyle, A.M. (1999) J Mol Biol 291(1): 15-27).
  • the flow rate was set to 5 ⁇ /minute, and the biotinylated C4-3 was injected at a concentration of 1 ⁇ over the streptavidin surface (SA sensor chip) for 15 minutes at 25°C.
  • the unbound aptamer was removed by treatment with 50 mM aqueous NaOH and the chip was primed before use.
  • TrkB protein solutions were sequentially injected over the sensor surface for 3 minutes at 15 ⁇ /min and 3 min dissociation time.
  • Six concentrations of TrkB protein were injected by serially diluting samples from 400 to 12.5 nM.
  • the selectivity studies were carried out by injecting 200 nM BSA and 200 nM TrkB protein over C4-3 aptamer and a control aptamer (4- IBB). The immobilization and injections for these control samples were performed under the same conditions described above. After each run, the surface was processed and analyzed to determine the binding constant for C4-3 aptamer. To correct for refractive index changes and instrument noise, the response of the control surface data was subtracted from the responses obtained from the reaction sur face sing BIA evaluation 4.1.
  • the D was calculated by global fitting of the six concentrations of TrkB protein assuming a constant density of C4-3 aptamer on surface.
  • a 1 1 binding aptamer measurements were aligned to C4-3 aptamer and TrkB for non-specific analysis.
  • the blots were incubated overnight with primary antibodies and subsequently with secondary antibodies (1 :5000) for 1 h at room temperature.
  • the antibodies and dilution used in this study are as follows: p-Trk (pY515), p-Trk (pY705/706), p-Src (Y416), p-Akt, p- Erk (1 :1000, Cell Signaling); TrkB (1:500, BD Transduction Laboratories; Bedford, MA); and ⁇ -actin (1 :10,000; Sigma, St. Louis, MO).
  • P-TrkB (pY816) (1 :1000) was kindly provided by Dr. Moses Chao (New York University).
  • the immunoblots were developed with enhanced chemiluminescense (ECL, Amersham; Piscataway, NJ). Equivalent amount of protein loaded in each lane was verified with immunoblotting with antibodies to TrkB or actin. Shown are representative results of immunoblotting from at least three independent experiments.
  • LDH lactate dehydrogenase
  • Hippocampal Interstitial Infusion Animals were handled according to National Institute of Health Guide for the Care and Use of the Laboratory Arrimals and approved by Duke University Animal Care and Welfare Committee. Adult mice were anesthetized with isoflurane (5% induction, 1.5% maintenance) and mounted in a stereotaxic apparatus. The skill was exposed, and a hole was drilled over the hippocampus (-2.5 mm AP, 2.2 mm ML).
  • KA Amygdala Infusion Continuous limbic and tonic-clonic seizures (status epilepticus) were induced in awake, adult male WT C57BL/6 mice weighing 20-25g by stereotaxic microinjection of 0.3 ⁇ g kainic acid (Sigma-Aldrich, St. Louis, MO) in a volume 0.25 ⁇ L of PBS (pH 7.4) at 0.056 ⁇ / ⁇ into the right basolateral amygdala nucleus through a guide cannula (Plastics One, Inc., Roanoke, VA).
  • the guide cannula was implanted in the right amygdala under pentobarbital anesthesia using >the following stereotaxic coordinates relative to bregama: AP, -0.94 mm; ML, +2.85 mm; and DV, -3.75mm (AP, anterior-posterior; ML, medio-lateral; DV, dorso-ventral); additionally, a bipolar EEG recording electrode was placed into the left dorsal hippocampus: AP, -2.00 mm; ML, - 1.60 mm; and DV, -1.53 mm.
  • TrkB-Specific Aptamers The mechanism of activation of TrkB has been well studied (see Figure 1).
  • the external signals that mediate activation of TrkB are neurotrophins including BDNF and NT4, 14 kDa proteins packed in dense-core vesicle of nerve terminals.
  • BDNF binds to the extracellular domain (ECD) of TrkB and induces receptor dimerization, leading to autophosphorylation of tyrosines within the intracellular domain and subsequent initiation of downstream signaling pathways.
  • ECD extracellular domain
  • tyrosines Once activated, surface TrkB is internalized into intracellular compartments.
  • RNAs bound to and subsequently internalized with murine TrkB expressing mammalian cells were isolated.
  • the initial step was to establish a mammalian cell line stably expressing TrkB.
  • HEK293 cells were transiently transfected with a plasmid expressing murine TrkB.
  • a single colony of G418- reistant and TrkB-expressing HEK cells was then selected.
  • the presence of TrkB on the HEK cell surface was verified with immunofluorescence (FIG 2B).
  • Incubation of TrkB activation as evidenced by increased phosphorylation of TrkB and Eri MAPK, a signaling protein downstream of TrkB ( Figure 2C).
  • TrkB-HEK cell line was subsequently used to select for RNA aptamers that bind TrkB.
  • the RNA library was "pre-cleared” by incubation with HEK cells lacldng TrkB ( Figure 2A and Materials and Methods). Following pre-clearing, the supernatant containing the RNA library was incubated with TrkB-expressing HEK cells and RNA internalized by these cells was recovered, amplified by PCR, and the procedure was repeated.
  • Selected Aptamers Activate TrkB Signaling in Cortical Neurons To test whether the selected aptamers can activate TrkB, the tyrosine phosphorylation of TrkB was monitored in aptamer-treated primary cultures of embryonic rat cortical neurons. Selected aptamers were prepared by in vitro transcription of gel-purified. Cortical neurons were incubated with vehicle, BDNF (10 ng.ml), or selected aptamers (200 nM) for 15 rnin. Cell lysates were subjected to SDS-PAGE followed by immunoblotting with p-TrkB antibodies.
  • BDNF induced an increase in phosphorylation of TrkB as well as downstream signaling molecules, Akt and Erk, providing a positive control for TrkB activation (Figure 4A).
  • aptamers including C4-2, C4-3, C4-6, C4-7, C5-2 and C5-3 was able to enhance the phosphorylation of TrkB, Akt, and Erk, indicating that these aptamers are TrlcB agonists ( Figure 4A and data not shown), thus indicating that the agonistic effects observed were not due to nonspecific effects of RNA application.
  • C4-3 Binds to the Extracellular Domain of TrkB: The observation that a subset of the selected aptamers activates TrlcB signaling in cultured neurons suggests that these aptamers bind directly to the ECD of TrkB. To test this possibility, an aptamer with agonist properties, C4-3, was selected for further study. Whereas the initial characterization of C4-3 utilized enzymatically synthesized RNA, subsequent characterization was carried out with chemically synthesized C4-3. Surface plasmon resonance was used to directly assess binding of C4-3 to the ECD of TrkB in a cell free context.
  • C4-3 Exerts Neuroprotective Effects on Cultured Cortical Neurons: Because reduced expression of BDNF is thought to contribute to death of CNS neurons in animal models of HD and AD, neuroprotective effects of C4-3 on cultured cortical neurons was evaluated. For this experiment, B27 growth supplement was withdrawn from healthy cultures of cortical neurons, which resulted in cell death that can be rescued by BDNF. Following B27 withdrawal from the culture for 48h, cell survival was assessed by measuring LDH release into the culture media (Lee, F.S. and Chao, M.V. (2001) Proc. Natl. Acad. Sci. USA 98:3555-3560). Neuronal cell death was induced upon B27 withdrawal (Figure 8).
  • RNA aptamers While small molecule drugs often suffer from unexpected and difficult to explain off-target effects, RNA aptamers exhibit specificities and affinities comparable to those of antibodies. Because RNA aptamers cannot diffuse across cell membranes, their non-specific potential is restricted (i.e., they have limited access to most intracellular compartments) compared to that of many small molecule drugs. The aptamer identification process is considerably less complex and expensive than that for small molecules because screening for aptamers is carried out with a single complex mixture whereas each member of a (usually vast) small molecule library must be screened individually. [0142] In contrast to antibodies, aptamers can be produced economically in large scale with chemical synthesis, are amenable to chemical modification and have low immunogenicity.
  • RNAs from a complex library that exhibits various desirable properties (in addition to target binding) in the course of aptamer development is another property that sets the aptamer platform apart from the development of other classes of therapeutics. Therefore, a number of aptamers for diverse targets of therapeutic interest have been developed, some of which have been evaluated and/or utilized in clinic (Keefe et al., 2010).
  • RTK-selective aptamers have led others to search for aptamers specific to particular RTKs, including HER3, RET, and Tie2 (see, e.g., Chen, et al. (2003) Proc Natl Acad Sci USA 100(16):9226-9231; Cerchia, L. et al. (2005) Plos Biol 3(4):el23; and White, R. et al. (2008) Angiogenesis 11(4):395-401).
  • Some of the aptamers that emerged from these selections exhibited antagonistic activity; however, aptamers with other signaling properties, e.g., agonist or partial agonist for a given RTK, were not described.
  • TrkB aptamer selection a cell based functional SELEX approach was established in which agonistic aptamers internalized to intracellular compartments after binding to the extracellular domain of TrkB were captured.
  • TrkB-HEK cell clones was screened by momtormg the TrkB protein contents with western blot analyses.
  • a TrkB-HEK cell line was established with a modest expression of exogenous TrkB in which application of BDNF resulted in a satisfactory degree of TrkB activation (FIG 2B).
  • RNAs selected with this approach was isolated and amplified, and the selected pool was sequenced with 454 high- throughput sequencing technology, which yielded thousands of distinct RNA sequences. This approach enabled identification of the sequences that were most prevalent at a relatively early round of selection. The majority of the 13 RNAs chosen from this group for characterization on a TrkB signaling assay exhibited TrkB agonistic activity in cultured rodent neurons ( Figure 4A), thus demonstrating the efficiency of the functional aptamer selection methodology.
  • C4-3 is a partial RNA agonist for TrkB: The discovery of diverse families of aptamers that bind TrkB permitted search for aptamers that possess agonistic activity, namely activation of TrkB in primary cultures of cortical neurons. The agonist activity of aptamer was monitored by Western blotting analysis of phosphorylation of TrkB (pTrkB), a surrogate measure of TrkB activation. A subset of selected RNAs was able to activate TrkB signaling in cultured neurons ( Figure 3), thereby validating the cell based functional assay for SELEX selection of RMA aptamers with agonist properties. C4-3 was chosen for more thorough characterization because it activates TrkB with high potency.
  • C4-3 functions as a selective partial agonist of TrkB in cortical neurons in vitro as evidence by: (1) C4-3 binds recombinant TrkB in a cell-free system and native TrkB expressed in mammalian cells; (2) C4-3 exhibits modest agonistic activity evident in its enhancement of pTrkB content at low nanomolar concentrations, potency consistent with its binding constant (3 ⁇ 4 of about 2nM) determined by surface plasma resonance assay in a cell free system; (3) its agonist activity notwithstanding, C4-3 is also able to inhibit BDNF-induced activation of its selective activation of TrkB because shRNA-mediated reduction of TrkB content in cortical neurons eliminated the biochemical evidence of TrkB activation by C4-3.
  • C4-3 the partial agonist activity of C4-3 proved sufficient to confer neuroprotective effects on mammalian cortical neurons maintained in vitro.
  • the presence of neuroprotective effects of C4-3 was examined in cultures in which neuronal cell death was induced by B27 withdrawal from cultured medium. Addition of exogenous BDNF to these cultures prevented cell death ( Figure 8), demonstrating that enhancing TrkB activation promotes neuronal survival under these conditions.
  • C4-3 (2 ⁇ ) but not scrambled aptamer potently reduced B27 withdrawal induced cell death in cultures ( Figure 8), thereby demonstrating a neuroprotective effect of C4-3 in vitro.
  • a partial agonist may tilt the balance of TrlcB signaling to a level with a beneficial effect yet prevent excessive activation of TrlcB.
  • partial agonists of neuronal receptors are clinically effective drugs including busporone (serotonin 5-HTiA receptor anxiety) and varenicline ( ⁇ 4 ⁇ 2 nicotinic cholinergic receptor for smoking cessation).
  • busporone serotonin 5-HTiA receptor anxiety
  • varenicline ⁇ 4 ⁇ 2 nicotinic cholinergic receptor for smoking cessation.
  • the functional cell-based SELEX approach described herein permitted identification of a selective partial agonist of TrkB with neuroprotective effects that lacked unwanted seizure-inducing actions.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un aptamère agoniste, C4-3, qui se lie au domaine extracellulaire du récepteur de kinase B liée à la tyrosine (TrkB) avec une affinité élevée, présente une puissante activité agoniste partielle TrkB et des effets neuroprotecteurs dans des neurones corticaux cultivés, et active TrkB lors d'une perfusion dans l'hippocampe. L'aptamère selon l'invention est également utile pour le traitement d'un ou de troubles neurologiques ou neuropsychiatriques. L'invention décrit également l'approche de sélection d'aptamère à base de cellule pour l'identification d'agonistes à base d'aptamère pour une diversité de récepteurs de signalisation de surface cellulaire.
PCT/US2012/067423 2011-12-02 2012-11-30 Aptamères d'acides nucléiques dirigés sur des récepteurs de surface et procédés d'utilisation Ceased WO2013082515A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161566120P 2011-12-02 2011-12-02
US61/566,120 2011-12-02

Publications (2)

Publication Number Publication Date
WO2013082515A2 true WO2013082515A2 (fr) 2013-06-06
WO2013082515A3 WO2013082515A3 (fr) 2013-08-15

Family

ID=48536242

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2012/067423 Ceased WO2013082515A2 (fr) 2011-12-02 2012-11-30 Aptamères d'acides nucléiques dirigés sur des récepteurs de surface et procédés d'utilisation

Country Status (1)

Country Link
WO (1) WO2013082515A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108265036A (zh) * 2018-01-26 2018-07-10 山东大学齐鲁医院 一种沉默t1蛋白的重组病毒及其构建方法与应用
CN110384712A (zh) * 2019-07-16 2019-10-29 南方医科大学 核酸适配子在制备治疗阿尔茨海默氏病药物中的应用
CN115074367A (zh) * 2022-06-22 2022-09-20 复旦大学附属眼耳鼻喉科医院 一组与脑源性神经营养因子高亲和力结合的核酸适配体及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1709178A4 (fr) * 2003-12-16 2008-01-09 Agency Science Tech & Res Procedes et composes permettant de modifier le degre de contamination microbienne d'un virus de l'hepatite
US20100076060A1 (en) * 2005-09-15 2010-03-25 Duke University Aptamers as agonists
EP2173377B1 (fr) * 2007-06-26 2017-11-29 University of Miami Protéine de fusion anticorps-endostatine et ses variants
WO2009012181A2 (fr) * 2007-07-13 2009-01-22 Ventana Medical Systems, Inc. Méthodes d'identification de réactifs de diagnostic

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108265036A (zh) * 2018-01-26 2018-07-10 山东大学齐鲁医院 一种沉默t1蛋白的重组病毒及其构建方法与应用
CN110384712A (zh) * 2019-07-16 2019-10-29 南方医科大学 核酸适配子在制备治疗阿尔茨海默氏病药物中的应用
CN115074367A (zh) * 2022-06-22 2022-09-20 复旦大学附属眼耳鼻喉科医院 一组与脑源性神经营养因子高亲和力结合的核酸适配体及其应用
CN115074367B (zh) * 2022-06-22 2024-12-13 复旦大学附属眼耳鼻喉科医院 一组与脑源性神经营养因子高亲和力结合的核酸适配体及其应用

Also Published As

Publication number Publication date
WO2013082515A3 (fr) 2013-08-15

Similar Documents

Publication Publication Date Title
JP7021320B2 (ja) 治療用分子を選択する方法
KR102021626B1 (ko) Ngf에 대한 압타머 및 그의 용도
Fernández et al. TLR4-binding DNA aptamers show a protective effect against acute stroke in animal models
JP2022532169A (ja) オリゴヌクレオチド組成物及びその使用方法
Huang et al. RNA aptamer-based functional ligands of the neurotrophin receptor, TrkB
US20250270555A1 (en) Organic compositions to treat beta-catenin-related diseases
JP7476199B2 (ja) Ｓｃｎ１ａ脳症の治療のためのｓｃｎ２ａを標的とするアンチセンスオリゴヌクレオチド
RU2699706C2 (ru) Вещества и способы модуляции заживления сухожилий
Wang et al. Role of corticotropin-releasing factor in cerebellar motor control and ataxia
JP2013539456A (ja) ＶＥＧＦＡをターゲティングするｓｉＲＮＡ及びｉｎｖｉｖｏ治療のための方法
WO2013186857A1 (fr) Aptamère de fgf2 et son utilisation
US10016424B2 (en) Method and pharmaceutical composition for use in the treatment of epilepsy
MX2014008456A (es) Acidos nucleicos que enlazan especificamente a cgrp.
US20160040126A1 (en) Regulation of differentiation into dopaminergic neurons by metalloprotease
JP2022531466A (ja) 遺伝性ニューロパチーおよび関連障害の処置および検出
WO2013082515A2 (fr) Aptamères d'acides nucléiques dirigés sur des récepteurs de surface et procédés d'utilisation
KR20150047513A (ko) 눈 증상의 치료 및/또는 예방을 위한 방법 및 조성물에서 sirna 및 이들의 용도
US20170355997A1 (en) Methods and compositions for treating or preventing pruritis
US20220372492A1 (en) Compounds for modulating fc-epsilon-ri-beta expression and uses thereof
EP2897633B1 (fr) Traitement de la douleur par inhibition de la déubiquitinase usp5
Twede et al. Conantokin-Br from Conus brettinghami and selectivity determinants for the NR2D subunit of the NMDA receptor
KR20160135819A (ko) Fgf2에 대한 압타머 및 그의 사용
WO2005092306A1 (fr) Procedes et compositions pour moduler la tolerance aux opioides et la douleur chronique
EP4605008A1 (fr) Ligands de l'intégrine alpha-v bêta-6 pour une distribution extra-hépatique
JP2025516677A (ja) Ｍａｐｔの発現を阻害するための組成物及び方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12852956

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 12852956

Country of ref document: EP

Kind code of ref document: A2