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WO2015075622A1 - Aptamères utilisés comme agents neuroprotecteurs contre la protéine basique de la myéline - Google Patents

Aptamères utilisés comme agents neuroprotecteurs contre la protéine basique de la myéline Download PDF

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Publication number
WO2015075622A1
WO2015075622A1 PCT/IB2014/066107 IB2014066107W WO2015075622A1 WO 2015075622 A1 WO2015075622 A1 WO 2015075622A1 IB 2014066107 W IB2014066107 W IB 2014066107W WO 2015075622 A1 WO2015075622 A1 WO 2015075622A1
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mbp
seq
aptamer
aptamers
fact
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Tomás Guido ROZENBLUM
Alfredo Daniel VITULLO
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FUNDACION CIENTIFICA FELIPE FIORELLINO
Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET
Inis Biotech LLC
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FUNDACION CIENTIFICA FELIPE FIORELLINO
Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET
Inis Biotech LLC
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    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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

  • the present invention provides aptamers against the Myelin Basic Protein (MBP), as well as the application thereof as neuroprotective agents in Multiple Sclerosis and as MBP detection agents .
  • MBP Myelin Basic Protein
  • Nucleic acid molecules with specific binding capacity to various target molecules are known in the art (antigens) which confers to them a huge potential for being used as therapeutic and diagnostic tools. These particular molecules are called aptamers and different manufacturing and isolation methods may be found in the literature.
  • Aptamers are single stranded nucleic acids (ssDNA or ssRNA) . Selection thereof is performed from libraries of oligonucleotides having a central region of variable size and randomized sequence and two flanking regions of known sequence which allow for PCR amplification. The central region is usually comprised between 30 and 60 nucleotides long, thus total length of the aptamer is 70-100 nucleotides.
  • Aptamers are able to bind to an antigen or target specifically and with high affinity, due to its spacial structure (Osborne, S. E. and Ellington, A. D., (1997); Nucleic Acid Selection and the Challenge of Combinatorial Chemistry . Chem Rev97(2) : 349- 370) .
  • many protein-recognition aptamers are also capable of interfering in protein biological function.
  • several techniques have been developed that facilitate the intracellular application of aptamers and its use as in vivo modulators of cell physiology. Highly specific agents acting against intracellular targets in the context of a living cell may be obtained by using these properties.
  • An aptamer in turn, may be selected in specifically designed experimental conditions, so that they are optimal for a specific diagnostic method (Jayasena, 1999) . Aptamers may be better stored for longer periods of time because, contrary to proteins, they are not subject to irreversible denaturation, rather they may be thermally renatured any time.
  • Stability they may withstand denaturing and renaturing processes. In turn, they may be modified during chemical synthesis for resisting the effect of various enzymes.
  • the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method is used for the selection of aptamers with higher affinity and specificity to the target molecule.
  • the population of aptamers is incubated together with a target molecule which may or may not be bound to a solid support and, interacting to a higher or lesser degree with the target protein according to their affinity.
  • Non- interacting molecules are removed and selected oligonucleotides are amplified by PCR and characterized by solid phase nucleic acid-sequencing, preserving a stock by introduction into bacteria by using cloning plasmids .
  • the individual aptamers are characterized according to their interaction with the target protein by different biochemical techniques such as surface plasmon magnetic resonance (SPR), ELISA, dot blot, or western blot.
  • the production of the selected aptamer is carried out by chemical synthesis, contrary to most of the molecules with amino acid chemistry acting in a similar fashion. Due to the interactions produced between bases along the chain, these molecules adopt tridimensional structures which allows them to bind stably and very specifically to their targets, which are small molecules through complex multimeric structures.
  • aptamers may be manufactured by chemical synthesis, it makes them suitable for production at large scale, bringing about a high reproducibility between production batches (Jayasena, 1999) .
  • RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J Biol Chem273(32) : 20556-20567), have been set as routine methods.
  • MS Multiple sclerosis
  • CNS central nervous system
  • MS is currently considered to be a CD4+ Thl-mediated autoimmune disease (1,2) .
  • This assumption is based on the cellular composition of brain and cerebrospinal fluid- infiltrating cells and also, on data obtained from the experimental (autoimmune) allergic encephalomyelitis model (EAE) (3) .
  • EAE is induced in susceptible rats and mice by the injection of myelin components promoting a CD4 + -mediated autoimmune disease which shares similarities with MS (1,3) .
  • MBP myelin basic protein
  • MBP Myelin Basic Protein
  • MBP-specific T cells may be isolated from MS patients and controls (4-10) .
  • MBP is the most widely studied myelin protein in MS. It is the second most abundant myelin protein after the proteolipid protein (PLP) .
  • MBP myelin stability is affected in the central nervous system.
  • T cells in MS patients are able to recognize MBP peptides exposed on antigen-presenting cells, which are capable of triggering an immune response against MBP.
  • the MS plaque is characterized by having focal areas of myelin destruction. These lesions are mostly seen in optic nerve, brain, spinal cord and white matter. In these plaques there is also lymphocyte infiltration in perivascular regions together with macrophages. MBP-specific CD4+ Thl cells have been characterized in MS patients.
  • the aptamer 3064 by Nastasijevic et al (2012) is 40 bases long and was injected into animals in the form of a multimeric complex, biotinylated and streptavidin-conj ugated, not being suitable for therapeutic administration.
  • Nastasijevic et al (2012) demonstrated certain remyelination, though no improvements were found in the cognitive and motor functions of the animals.
  • aptamers are provided that specifically recognize the Myelin Basic Protein (MBP) and have a length of less than 100 nucleotides.
  • MBP Myelin Basic Protein
  • the aptamers of the invention may be single stranded DNA or RNA.
  • aptamers or variants thereof comprise the sequence 5' GCGTCGATTGCCATGGGTTG 3' (SEQ ID N° 1) on the 5' end, and the sequence 5' CACGCAGTGAGCTCCCCTGGACC 3' (SEQ ID N° 2) on its 3 ' end .
  • the aptamers or variants thereof according to the present invention preferably have a length of more than 60 nucleotides and less than 93 nucleotides, and more preferably, a length of more than 60 nucleotides and less than 70 nucleotides.
  • the aptamer or variants thereof is selected from:
  • nucleotides of the aptamers of the invention may be modified with any one of the following: phosphorothioate, Locked Nucleic Acids (LNA), Morpholino phosphorodiamidate , N3'-P5' phosphoramidate, 2 ' -C-allyl uridine, 2'-fluoro nucleotides, 2' -amino nucleotides.
  • LNA Locked Nucleic Acids
  • Morpholino phosphorodiamidate N3'-P5' phosphoramidate
  • 2 ' -C-allyl uridine 2'-fluoro nucleotides
  • 2' -amino nucleotides 2' -amino nucleotides.
  • the present invention also provides pharmaceutically suitable aptamer salts, which may be manufactured by known methods.
  • the aptamers of the present invention are also useful as therapeutic agents for the treatment and prevention of all those MBP-related diseases, in particular, demyelinating and dysmyelinating diseases, in particular, it relates to Multiple Sclerosis (MS) .
  • MS Multiple Sclerosis
  • the invention relates to the aptamers of the invention or mixtures of several aptamers for its use in medicine, as well as to pharmaceutical compositions comprising at least an aptamer of the invention and derivatives thereof, which homologies are 40% or less and, optionally, one or more pharmaceutically acceptable supports, excipients or solvents .
  • the present invention also relates to the use of nucleic acids, according to the invention, as a diagnostic tool.
  • Fig. 1A Binding activity of aptamers after 15 cycles. Aptamer binding was detected by a dot-blotting after 15 SELEX selection rounds. l]ig of MBP was placed on a nitrocellulose membrane and incubated with equal nanomolar amounts of biotinylated aptamers in selection buffer containing 0.1 mg/ml yeast tRNA. The selected library against MBP is MBP C15; Cll is another aptamer group selected against another MBP- unrelated target molecule. Bound aptamers were detected with an anti-biotin alkaline phosphatase-conjugated antibody.
  • Fig. IB Binding activity of aptamers selected against MBP.
  • MBPcl3 and MBPcl9 are unique MBP-specific clones. The same micromolar amount of MBP and AGP were placed on the nitrocellulose membrane and incubated with equal nanomolar amounts of biotinylated aptamers MBPcl3, MBPcl9 and a non- selected library.
  • Fig. 1C Role of attached biotin in the MBPcl3 binding activity to MBP.
  • l]ig of MBP was placed on a nitrocellulose membrane and incubated with equal nanomolar amounts of biotinylated aptamers or biotin alone.
  • MBPcl3 is clone 3 of the selected aptamers.
  • "Only biotin” is a membrane which was incubated with free biotin.
  • MBPcl3 DNAsel is clone 3 of the aptamer treated with the enzyme and MBPcl3 without DNAsel is the aptamer with the enzyme buffer solution.
  • Fig. 2 Sequence alignment of the two more representative clones of the enriched library selected against MBP . The sequence corresponding to the primers' binding region is underlined. Bold letters show the differences between both clones .
  • Fig. 3 MBP binding activity of MBPcl3 as compared to shuffled sequence-MBPcl3.
  • Fig. 4A Prediction of MBPcl9 secondary structure and possible binding active sites to MBP
  • Fig. 4B Prediction of MBPcl3 secondary structure and possible binding active site to MBP
  • Fig. 4C Prediction of MBPcl3 secondary structure and possible binding active site to MBP
  • MBPcl9 AF includes stem 5, loop A and F; MBPcl3 BC includes stem 8 and loops B and C; MBPcl3 includes stem 7 with loops A and E.
  • Fig. 6A Binding activity of MBPcl3 and aptamer 3064 to MBP.
  • Fig. 6B Binding activity of MBPcl3 and aptamer 3064 to MBP in a complex protein mixture. Mass units on the right side of aptamer 's name depicts the amount of MBP present in the complex protein mixture (1, 0.5 or 0 ⁇ g) .
  • Fig. 7A Binding activity of MBPcl3 and an anti-MBP polyclonal antibody to MBP.
  • Fig. 7B Binding activity of MBPcl3 and an anti-MBP polyclonal antibody to MBP in a complex protein mixture. Mass units on the right side of aptamer 's or IgG name depicts the amount of MBP present in the complex protein mixture (1 or 0.5 ⁇ g) .
  • Fig. 7C MBPcl3 and an anti-MBP polyclonal antibody competition for MBP . Columns: First column, MBP was incubated with the anti-MBP antibody, washed and then incubated with aptamer MBPcl3. Second column, MBP was incubated only with anti-MBP. Third column, MBP was incubated with MBPcl3, washed and then incubated with anti-MBP. Fourth column, anti-MBP and MBPcl3 were incubated together. Fifth column, anti-MBP was incubated without MBP.
  • Fig. 8A Binding activity of MBPC13 and a version thereof with nucleotides modified with phosphorothioate (MBPcl PS) to MBP.
  • Lib is the binding activity of the non-selected original library .
  • Fig. 8B Binding activity of MBPC13 and a phosphorothioate version (MBPcl PS) to MBP using a brain myelin extract as a MBP source.
  • Lib is the binding activity of the non-selected original library.
  • Fig. 9 Localization of MBPC13 in histological mouse brain sections. Serial mouse brain sections were used for assessing the localization of an anti-myelin antibody (A) and with aptamer MBPC13 (B) . MBPC13 was detected in myelin-rich areas such as corpus callosum, cerebral peduncle, thalamic radiation, fasciculus retroflexus, and premammilary nucleus. Both the MBP-specific antibody and MBPC13 were localized to similar brain areas.
  • A anti-myelin antibody
  • B aptamer MBPC13
  • the aptamers of the present invention are single stranded nucleic acid molecules and include DNA and RNA molecules, as well as variants thereof which have been modified with the aim of improving the molecules' resistance to body nucleases and pharmacokinetics.
  • modifications include nucleotide modifications, phosphodiester bond modifications, as well as polyethylene glycol conjugates.
  • the aptamers share with the natural DNA a very short life-time in blood, estimated to be of 1 to 2 minutes. This short life-time is sometimes attributed to the serum nuclease.
  • the serum nuclease gives truncated aptamer DNA fragments as a result of its activity, lacking the affinity for the target protein .
  • nucleic acids are understood as polymeric molecules which, in the case of RNA, are composed of the nucleotides adenosine (A), cytidine (C), uridine (U) , guanosine (G) and, in the case of DNA, are composed of deoxyadenosine (A), deoxycytidine (C), deoxyguanosine (G) and thymidine (T) .
  • nucleotides may display at least one of the following modifications: 2'-deoxy, 2'-fluoro, 2'-chloro, 2'-bromo, 2'-iodo, 2' -amino (preferably unsubstituted or mono- or disubstituted) , 2' -mono-, di- or tri-halo-methyl , 2'-0-alkyl, 2 ' -O-halo-substituted alkyl, 2'- alkyl, azido, phosphorothioate, sulfhydryl, methylphosphonate, fluorescein, rhodamine, pyrene, biotin, xanthine, hypoxanthine , 2 , 6-diaminopurine, 2-hydroxy-6-mercaptopurine, polyethylene glycol modifications and for the sulfur of the pyrimidine bases at position 6 and the halogen at position 5, Cl-5 alkyl, abasic linkers, 3
  • nucleotide analogs For an aptamer to retain affinity for its target protein, the same nucleotide analogs should be used as those used in the selection experiment for the synthesis of the aptamer for practical use.
  • the introduction of nucleotide analogues that override the contacts between the aptamer and the target protein should be avoided, since it would result in a decrease in binding affinity.
  • the affinity of aptamers for their target proteins is typically in the nanomolar range, but can be as low as the picomolar range. That is, Kd is typically 1 pM to 500 nM, more typically from 1 pM to 100 nM. Aptamers that have an affinity of Kd in the range of 1 pM to 10 nM are also useful.
  • Aptamers will typically have a length of 10 to 200 nucleotides, more typically less than 100.
  • the aptamers of the present invention have a preferred length of less than 100 nucleotides, being preferably greater than 60 and less than 93 nucleotides, and still most preferably greater than 60 and less than 70.
  • Nucleic acids according to the present invention may be manufactured in a simple manner, for example, by conventional chemical synthesis using a DNA / RNA solid phase synthesizer, without representing a problem for the expert in the art.
  • the corresponding protocols and devices are known to the skilled person in the art and are of routine use.
  • the product may be purified by size selection methods or by chromatographic methods .
  • Aptamer sequences may be chosen as a desired sequence, or randomized- or partially randomized- sequence populations may be made, and then be selected for their specific binding to myelin basic protein. Any of the tests typically known in the art for assessing nucleic acid-protein binding may be used, for example, Southwestern blotting using either labeled oligonucleotides or labeled protein as a probe.
  • SELEX (TM) Systematic Evolution of Ligands by Exponential Enrichment
  • the SELEX(TM) process is a process for the in vitro evolution of nucleic acid molecules having high specific binding to target molecules and is described, for example, in Patent Application US Serial No. 07/536,428 filed June 11, 1990, now abandoned, US patent No. 5,475,096 titled “Nucleic acid ligands” and US patent No. 5,270,163 (see also WO 91/19813) titled "Nucleic acid ligands”.
  • Each nucleic acid ligand identified in the SELEX (TM), that is, each aptamer, is a specific ligand for a given target compound or molecule.
  • the SELEX (TM) process is based on the unique knowledge that nucleic acids have sufficient capacity to form a variety of bi- and tri-dimensional structure and sufficient chemical versatility available within its monomers in order to act as ligands (i.e., they form specific binding pairs) with practically any chemical compound, both monomeric and polymeric. Molecules of any size and composition may serve as targets .
  • SELEX is based, as a starting point, in a large library or single stranded oligonucleotide set which comprise randomized sequences.
  • Oligonucleotides may be DNA, RNA, DNA/RNA hybrids, either modified or non-modified.
  • the set comprises 100% of randomized or partially randomized oligonucleotides.
  • the set comprises randomized or partially randomized oligonucleotides containing at least one fixed and/or conserved sequence incorporated within the randomized sequence.
  • the set comprises randomized or partially randomized oligonucleotides containing at least one fixed and/or conserved sequence on its 5' and/or 3' end, which may comprise a sequence shared by all the molecules of the oligonucleotide set.
  • Fixed sequences are sequences which are common to nucleotides in the set, which is incorporated for a preselected purpose, such as CpG motifs, annealing sites for PCR primers, promoter sequences for RNA polymerases (for example, T3, T4, T7 and SP6), restriction sites or homopolymeric sequences such as portions of poly A or poly T, catalytic cores, sites for selective binding to affinity columns and other sequences for facilitating cloning and/or sequencing of an oligonucleotide of interest.
  • conserveed sequences are sequences, different to the fixed sequences previously described, shared by several aptamers which bind the same target.
  • the oligonucleotides of the set preferably include a portion of randomized sequence, besides the fixed sequences required for efficient amplification.
  • the oligonucleotides of the starting set contain fixed sequences of the 5'- and 3' -end flanking an internal region of 30-50 random nucleotides.
  • Random nucleotides may be produced by several ways including chemical synthesis and size selection from the cellular nucleic acids cleaved at random. Sequence variation in the randomized region of the test nucleic acids may also be modified by means of the introduction or increase of sequences by mutagenesis before or during the selection/amplification iterations .
  • the portion of the randomized sequence of the oligonucleotide may be of any length and may comprise ribonucleotides and/or deoxyribonucleotides and may include modified nucleotides, or non-natural or nucleotide analogs. See, for instance, US patent No. 5,958,691; US patent No. 5,660,985; US patent No. 5,958,691; US patent No. 5,698,687; US patent No. 5,817,635; US patent No. 5,672,695 and PCT publication WO 92/07065. Randomized oligonucleotides may be synthesized by using oligonucleotide synthesis techniques in solid phase, well known in the art.
  • Randomized oligonucleotides may also be synthesized by using dissolution-free processes such as the triester synthesis processes. See, for instance, Sood et.al., Nucl. Acid Res. 4:2557 (1977) and Hirose et.al., Tet. Lett., 28:2449 (1978) .
  • Typical synthesis carried out in DNA synthesis automated equipment yield 10 14 -10 16 individual molecules, a number sufficient for most of the SELEXTM assays. Sufficiently large regions of the randomized sequence in the sequence design increases the chances that each synthesized molecule likely represents a single sequence.
  • the starting oligonucleotide library may be generated by automated chemical synthesis in a DNA synthesizer.
  • a DNA synthesizer For the synthesis of randomized sequences, mixtures of four nucleotides are added to each nucleotide addition step during the synthesis process, allowing the random incorporation of the nucleotides.
  • the randomized oligonucleotides comprise complete randomized sequences; however, in other embodiments the randomized oligonucleotides may comprise extensions of nonrandomized or partially randomized sequences. Partially randomized sequences may be created by adding the four nucleotides at different molar ratios in each addition step.
  • the starting oligonucleotide library may be either RNA or DNA, or RNA or DNA with modified nucleotides.
  • an RNA library is usually generated by synthesizing a DNA library, optionally amplifying by PCR, then transcribing the DNA library in vitro by using RNA polymerase T7 or modified RNA polymerases T7, and purifying the transcribed library. Then, the RNA or DNA library is mixed with the target in conditions favorable for the binding, and it is subject to step-wise binding, separation and amplification iterations, using the same general selection scheme, for achieving practically any desired affinity and binding selectivity criteria.
  • the SELEX (TM) process includes the steps of: (a) contacting the mixture with the target in conditions favorable for the binding; (b) separating unbound nucleic acids from those nucleic acids which have specifically bound to target molecules; (c) dissociating the nucleic acid- target complexes; (d) amplifying the nucleic acids dissociated from the nucleic acid-target complexes in order to give an nucleic acid ligand-enriched mixture; and (e) reiterating the steps of binding, separation, dissociation and amplification throughout as may cycles as desired to yield highly specific nucleic acid ligands with high affinity for the target molecule.
  • the SELEX(TM) process also comprises the steps of: (i) reverse transcription of the dissociated nucleic acids of the nucleic acid-target complexes before the amplification in step (d) ; and (ii) transcription of the nucleic acids amplified in step (d) before starting the process again.
  • a mixture of nucleic acids comprising, for example, a segment of 20 nucleotides at random by have 1.09 12 candidate possibilities. Those having the higher affinity (lower dissociation constants) for the target are the ones more likely to bind the target.
  • a second mixture of nucleic acids is generated, which is enriched in the candidates with higher binding affinity. Additional selection rounds progressively favor the best ligands until the resulting nucleic acid mixture is predominantly composed of only one or some sequences. Then, this may be cloned, sequenced and tested individually for the binding affinity as pure ligands or aptamers.
  • the selection and amplification cycles are repeated until a desired object is achieved. For the most general case, selection/amplification continues until a significant improvement in the binding strength is achieved by cycle repetition.
  • the process is usually used for sampling approximately 10 14 species of different nucleic acids, but may be used for sampling up to approximately 10 18 species of different nucleic acids.
  • the nucleic acid aptamer molecules are selected in a process of 5 to 20 cycles.
  • US patent No. 5,707,796 discloses the use of SELEX(TM) together with gel electrophoresis for selection of nucleic acid molecules with specific structural characteristics, such as bent DNA.
  • US patent nro. 5,763,177 discloses SELEX ( TM) -based processes for selection of nucleic acid ligands containing photoreactive groups which bind and/or photocrosslink to and/or photoinactivate a target molecule.
  • the object of the present invention is, therefore, to provide MBP-binding aptamers suitable for therapeutic use, which display a suitable specificity and biodistribution in order to obtain remyelination together with enhanced cognitive and motor functions in animals.
  • aptamers which specifically recognize the Myelin Basic Protein (MBP) and display a length of less than 100 nucleotides, of the present invention. In fact, its reduced size guarantees its biodistribution.
  • MBP Myelin Basic Protein
  • aptamers of the invention may be single stranded DNA or RNA.
  • aptamers or variants thereof are provided which comprise the sequence 5 ' GCGTCGATTGCCATGGGTTG (SEQ D N ° 1), or its RNA
  • aptamers or variants thereof are provided which comprise the sequence 5 ' CACGCAGTGAGCTCCCCTGGACC (SEQ ID N° 2), or its RNA
  • the aptamers or variants thereof according to the present invention preferably have a length of more than 60 nucleotides and less than 93 nucleotides, and more preferably, a length of more than 60 nucleotides and less than 70 nucleotides.
  • aptamers or variants thereof according to the present invention comprise the following preferred sequences:
  • the aptamer or variants thereof is selected from:
  • the aptamer or variants thereof is : 5 ' GCGTCGATTGCCATGGGTTGGGTCCGCGCAGTGAGCTCTCCTGGACCACGCAGTGAGCTC CCCTGGACC (MBP_C13) (SEQ ID N°3) .
  • the aptamer or variants thereof is :
  • the invention is subject to particular modifications thus providing a molecule with the same features but with some nucleotide modifications which confer higher serum stability to the molecule.
  • the same molecule may be used for conjugation to other chemical groups adding functionality such as for example, facilitating the translocation of the aptamer through the blood-brain barrier.
  • the modified nucleotides may be any one of the following: phosphorothioate, Locked Nucleic Acids (LNA), Morpholino phosphorodiamidate, N3'-P5' phosphoramidate, 2'-C-allyl uridine, 2'-fluoro nucleotides, 2' -amino nucleotides.
  • Pharmaceutically suitable salts may be also obtained from the aptamers according to the present invention, which may be manufactured using known methods such as dissolving the compounds according to the present invention, in the corresponding aqueous buffer solutions or in H 2 0 and subsequently lyophilizing them.
  • Metallic salts may be obtained by dissolving the compounds according to the present invention, in solutions containing the corresponding ion and subsequently isolating the compound using HPLC or gel filtration methods.
  • aptamers are not only capable of binding a target molecule but also may effect structural alterations therein which may lead to the loss of activity in the target molecules or may interfere with the interaction of the target protein with other proteins in the cells.
  • the aptamers of the present invention are also useful as therapeutic agents for the treatment and prevention of all those MBP-related diseases, in particular, autoimmune or demyelinating diseases of the nervous system, more specifically, demyelinating autoimmune diseases.
  • MBP-related diseases may be: encephalomyelitis, including viral and allergic encephalomyelitis, hypoxia, trauma leukodystrophy neoplasia, Wernicke disease, Guillen- Barre syndrome and central nervous system lupus, and demyelinating diseases of the central nervous system (such as, multiple sclerosis) .
  • demyelinating diseases may be: Clinically isolated syndrome, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Balo's concentric sclerosis, Marburg's disease, isolated acute myelitis, multiple sclerosis, optic neuromyelitis, opticospinal multiple sclerosis, isolated recurrent optic neuritis, chronic relapsing inflammatory optic neuropathy, recurrent acute myelitis, delayed post-anoxic encephalopathy, osmotic myelinolysis .
  • the aptamers of the present invention are especially suitable for the treatment and/or prevention (or delay in the development) of multiple sclerosis (MS) .
  • the invention relates to aptamers of the invention or mixtures thereof, for its use in medicine, as well as to pharmaceutical compositions comprising at least an aptamer of the invention and, optionally, one or more pharmaceutically acceptable supports, excipients or solvents.
  • the specific medication and posology are dependent upon several factors including activity of the specific compounds used, age of the patient, body weight, overall health state, sex, nutrition, time of administration, administration method, excretion rate, interaction with other medicines, and severity of the disease for which the therapy is applied. These shall be determined by a physician based on the mentioned factors .
  • the compounds and combinations of compounds of the invention may be formulated together with an excipient which is acceptable from a pharmaceutical point of view.
  • Preferred excipients for use in the present invention include sugars, starches, celluloses, gums and proteins.
  • the pharmaceutical composition of the invention shall be formulated in a pharmaceutical administration solid form (e.g., tablets, capsules, pills, granules, suppositories, etc.) or liquid form (e.g., solutions, suspensions, emulsions, etc.) .
  • compositions of the invention may be administered through any route, including, without limitation, oral, intravenous, intramuscular, intraarterial, intramedular, intrathecal, intraventricular, transdermic, subcutaneous, intraperitoneal, intranasal, enteric, topical, sublingual or rectal routes.
  • routes including, without limitation, oral, intravenous, intramuscular, intraarterial, intramedular, intrathecal, intraventricular, transdermic, subcutaneous, intraperitoneal, intranasal, enteric, topical, sublingual or rectal routes.
  • compositions which include at least one of the aptamers according to the invention.
  • the present invention also relates to the use of nucleic acids, according to the invention, as a diagnostic tool.
  • labeled nucleic acids may be used. Labelling may be accomplished for example by a fluorescent dye, enzyme, antibody or radionuclide. Corresponding detection methods are known to those skilled in the art.
  • an aptamer according to the invention is a component part of a kit, together with, for example, negative and positive controls .
  • the expert perfectly knows that the corresponding aptamers may be applied, in principle, as antibodies for example, in ELISA applications, chromatographic methods and diagnostic/tracing procedures.
  • Mouse myelin basic protein, Bovine Serum Albumin fraction V, alkaline phosphatase-conj ugated monoclonal anti- biotin, human alpha acid glycoprotein and anti-MBP polyclonal antibodies were purchased from Sigma Aldrich.
  • Yeast tRNA was purchased from Biodynamics. Taq Polymerase and dideoxynucleotides were obtained from Fermentas .
  • the colorimetric substrates of the alkaline phosphatase (nitroblue tetrazolium chloride and bromo-chloro-indolyl [NBT/BCIP]) were purchased from Promega.
  • HRP-conj ugated avidin was purchased from Pierce.
  • Alkaline phosphatase-conjugated streptavidin was purchased from Promega. High binding capacity Microlon ELISA plates were obtained from Greiner Bio-One. The oligonucleotides were synthesized by Integrated DNA Technologies . Nitrocellulose HybondTM ECLTM membranes were obtained from Amersham Biosciences. Salmon sperm DNA was purchased from Life Technologies . All other reagents and chemicals were of the highest purity available and were obtained from commercial sources. SELEX.
  • the starting single stranded DNA (ssDNA) library was designed to contain a 35-nucleotide randomized sequence region, flanked by constant primer annealing regions (5'- GCGTCGATTGCCATGGGTTG (N) 35 CACGCAGTGAGCTCCCCTGGACC-3') -
  • a biased molar volume (30% A, 30% C, 20% G and 20% T) was used for the synthesis of the four phosphoramidites was used to compensate for their incorporation efficiencies and to generate a truly randomized region.
  • the first round of selection was initiated with 0.5 nmol (approximately 3xl0 14 molecules) of ssDNA.
  • ssDNAs Prior to selections, ssDNAs were dissolved in a buffer solution (BS; 20mM Tris-HCl pH 7.6, 150mM NaCl, 5mM MgCl 2 ), denatured at 95°C for 5 minutes and cooled on ice for another 5 minutes. Affinity selections were done as follows: MBP was immobilized onto a nitrocellulose membrane disc and blocked in blocking buffer (BS containing 2% BSA, bovine serum albumin) for 60 minutes. After several washes with BS, MBP was incubated with the ssDNA at 37 °C for a fixed period of time.
  • BS buffer solution
  • BSA bovine serum albumin
  • Counter-selection of aptamers was performed by incubating the ssDNA library with nitrocellulose membrane discs that had been blocked with blocking buffer for 60 minutes at SELEX cycles 2, 3, 4, 5, 7, 9, 11, and 12. Molar ratio of MBP to aptamers, as well as washing stringency, and incubation time were modified over the SELEX cycles (table 1) .
  • aptamers bound to the MBP were eluted in distilled water by heating the compound at 95°C for 10 minutes.
  • Asymmetric Polymerase Chain Reaction (asymmetric PCR) was performed with the eluted aptamers in 100 ⁇ -reactions containing: 2 ⁇ forward primer 5 > -GCGTCGATTGCCATGGGTTG-3' (SEQ ID N°l); 2 ⁇ reverse primer 5' -GGTCCAGGGGAGCTCACTGCGTG-3' ( SEQ ID N°18); 200 ⁇ of each of the triphosphate deoxyribonucleotide (dNTP); lOmM Tris-HCl (pH 8.8 at 25°C), 50mM KC1, 0.08% (v/v) Nonidet P40, 0.8M MgCl 2 .
  • the thermal cycle consisted of one cycle at 95°C for 10 minutes and cycles of 95°C for 30 seconds, at 58°C for 40 seconds and 72°C for 40 seconds.
  • the number of PCR cycles was estimated empirically in order to avoid the amplification of PCR products of incorrect sizes.
  • single stranded DNA was precipitated with ethanol and a pellet was collected by centrifugation . The pellet was vacuum dried and resuspended in distilled water for use in the next selection round as a template .
  • Biotinylated aptamers were generated by asymmetric PCR with a biotinylated forward primer 5 ' or by solid phase synthesis. Aptamers were heated to 95°C for 5 minutes and then chilled on ice for another 5 minutes. Pure MBP protein was applied to a nitrocellulose membrane with a pore size of 0,45 ⁇ and was dried in air. The membranes were blocked with a blocking buffer or Tris buffered saline (TBS; 20 mM Tris-HCl pH 7.5, 500 mM NaCl) with 2% bovine serum albumin for one hour at room temperature.
  • TBS Tris buffered saline
  • the membranes were then washed three times with BS or TBS, and incubated with 0.014 nmoles aptamers in BS or TBS containing 0.1 mg/ml yeast RNAt for 2 hours at 37°C. Then, the membranes were washed three times with BS or TBS and were incubated for 1 hour at room temperature with an anti-biotin monoclonal antibody conjugated to alkaline phosphatase in BS or TBS containing 2% bovine serum albumin. The membranes were washed three times and the biotinylated aptamers were detected with the colorimetric substrates of the alkaline phosphatase (NBT/BCIP) .
  • NBT/BCIP colorimetric substrates of the alkaline phosphatase
  • Binding assays A Microlon high capacity ELISA plate is coated overnight with MBP in lOOul 0.05 M sodium carbonate pH 9.6. The wells were washed three times with phosphate buffer saline (PBS) containing Tween 20 (PBS-T; IMm KH 2 P0 4 , lOmM Na 2 HP0 4 , 137mM NaCl, 2.7mM KC1, 0.05% Tween 20) and were blocked with PBS-T containing 3% bovine serum albumin for 1 hour. Then, the wells were washed three times with BS and were incubated with aptamers in BS in the presence of 0.1 mg/ml yeast tRNA or salmon sperm DNA.
  • PBS-T phosphate buffer saline
  • aptamers Before the addition of aptamers, they were heated at 95°C for 5 minutes and chilled on ice for another 5 minutes. The wells were washed three times with BS and HRP-conj ugated avidin was added in BS containing 2% bovine serum albumin for 1 hour. Then, the wells were washed three times with BS and TMB (3,3', 5,5'- tetramethylbenzydine ) was used for the HRP substrate. Absorbance was measured at 450nm with a Biotek® ⁇ ) ⁇ 3 ⁇ TM microplate spectrophotometer.
  • Binding assay with protein mixture Three plates with HeLa (ATCC®) cells grown to confluence were washed twice with PBS and lysed in RIPA buffer solution (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 NP-40 1%, 0.5% sodium deoxycholate, 0.1% SDS, 0.2mM phenylmethylsulfonyl fluoride, 10 nM leupeptin and 10 pg/ml aprotinin) . The lysates were centrifuged at 15,000 xg in a table centrifuge at 4°C for 15 minutes and were collected in a single tube.
  • RIPA buffer solution 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 NP-40 1%, 0.5% sodium deoxycholate, 0.1% SDS, 0.2mM phenylmethylsulfonyl fluoride, 10 nM leupeptin and 10 pg/ml aprotinin
  • Microlon ELISA plates were prepared as described above and 75 ⁇ 1 of cell lysates were added for 1 hour to each well that previously had the MBP protein. After three washes with PBS-T, the wells containing the MBP protein and cell lysates with PBS-T containing 3% bovine serum albumin were blocked for 1 hour. Aptamers were added and detection continued as described above (Binding Assays) .
  • mice were fixed in paraformaldehyde solution (PFA) and embedded in paraffin. Paraffin-embedded tissue was sectioned into 5 ⁇ m-thick slices. Histochemical identification of myelin was performed as follows: Sections of mouse brain tissue were deparaffinized in xylene, rehydrated in alcohol of decreasing graduations, washed in distilled water for 5 min . and reserved in IX BS until its use.
  • PFA paraformaldehyde solution
  • the tissue was incubated in PBS containing 4% PFA and 0.3% triton for 1 min., then washed and incubated with 5mM MBPC13 for 2 hours at 37 °C in IX BS containing 2% BSA. Color development was carried out in the commercial reagent kit SIGMA Magenta and assessment was performed on a Nikon Eclipse E200 microscope.
  • tissue sections were incubated in PBS containing 4% PFA and 0.3% triton for 1 min. Then, they were washed and treated with H 2 0 2 0.3% in water for quenching the activity of endogenous peroxidases. Then, the tissue sections were washed and blocked with PBS containing 2% BSA for 30min. Then they were washed and incubated with the rabbit anti-MBP antibody in PBS containing 1% BSA for 1 h. at room temperature. Then, the sections were washed and incubated with an anti-rabbit IgG HRP-conj ugated antibody for 1 h. in PBS containing 1% BSA. Color development was carried out with the commercial reagent Vectastain elite ABC kit (rabbit IgG) from Vector Laboratories and assessment was performed in a Nikon Eclipse E200 microscope .
  • Single-stranded DNA molecules exhibiting high affinity for mouse MBP were obtained by an in vitro aptamer selection process, named SELEX.
  • SELEX Single-stranded DNA molecules exhibiting high affinity for mouse MBP were obtained by an in vitro aptamer selection process, named SELEX.
  • Table 1 The SELEX cycle was started with a library of ⁇ 3 10 14 distinct ssDNA molecules. Aptamers obtained at the 15th cycle (MBP C15) were analyzed by dot-blotting (Figure la) .
  • Figure la shows the MBP-binding activity of the pool of specific aptamers compared to the activity of another pool of aptamers that had been selected to an unrelated protein. After 15 SELEX rounds, the aptamer pool was cloned and 15 clones were sequenced.
  • the unselected aptamer pool also showed a strong interaction with dots of MBP suggesting that MBP bound to nucleic acids irrespective of their sequence due to its highly positive net charge at physiological pH; however, it is also possible that the binding activity resides in part within the annealing region of the reverse primer corresponding to the aptamer.
  • the same test on an ELISA plate format exhibits a weak binding of the unselected library. Sequence analysis of both aptamer clones revealed a highly conserved nucleotide sequence between the randomized region and the annealing region of the reverse primer of the aptamers.
  • Biotinylated aptamers were used in the Dot Blotting and ELISA analysis used in the above assays; therefore, it is important to verigy whether biotin participates in the binding activity of the selected aptamers, thus MBP was placed on nitrocellulose membranes, incubated with biotinylated MBPcl3, biotin or biotinylated MBPcl3 subjected to digestion with DNAse (Fig. 1C) . Clearly, Fig. 1C shows that biotin does not bind to MBP.
  • MBPcl9 and MBPcl3 were Structural analysis of MBPcl9 and MBPcl3 was performed with Mfold (12) to predict the most likely single-stranded DNA secondary structures (Fig. 4 A, B y C) .
  • MBPcl9 structure displayed an inverted L-shape (Fig. 4A) with two long stems, each followed by a pair of loops and several terminal unpaired nucleotides.
  • two optimal structures are shown for MBPcl3.
  • One of these structures shows the inverted L-shaped figure, shared with MBPcl9 (Fig. 4B) .
  • the second structure (Fig. 4C) contains a higher number of unpaired nucleotides.
  • an aptamer named 3064 was raised from a suspension of crude murine myelin and it was claimed that it is specific against MBP isoforms (13) . Therefore, the activity of MBPcl3 aptamer was compared in different environments against the known aptamer 3064 ( Figure 6 A) . It should be noted that aptamer 3064 showed a slightly higher activity when a pure protein of MBP was used; however, a larger standard deviation for this aptamer was observed in all trials. Furthermore, when the MBP protein was mixed with a complex mixture of proteins, 3064 aptamer performance was poor (Figure 6B) . Different amounts of MBP were fixed to Microlon ELISA plates that had a hydrophobic surface.
  • HeLa cell lysate was used as a blocking reagent followed by a blocking solution of bovine serum albumin.
  • the plate wells with less amount of MBP let more surface area for fixing HeLa cell lysates and bovine serum albumin protein.
  • the aptamer 3064 exhibited a binding pattern not related to the concentration of MBP, suggesting increased non-specific binding activity (Figure 6B) .
  • MBPcl3 binding activity was also compared with a commercial anti-MBP polyclonal antibody.
  • equal molar concentrations of MBPcl3 and anti-MBP antibody were incubated with con 0.5]ig of MBP that was previously fixed to a Microlon ELISA plate ( Figure 7) .
  • MBPcl3 binds to MBP with a nearly 5-fold higher activity than the antibody.
  • MBPcl3 was also compared with anti-MBP antibody in a complex mixture of proteins ( Figure 7B) . The assay was performed as described for the aptamer 3064.
  • Multiple sclerosis is a disease where autoimmune antibodies play a key role.
  • MBP recognition by the autoantibodies induce lymphocyte recruitment to MBP sites and could be involved in the degradation of the myelin protein.
  • a competition assay ( Figure 7C) was performed.
  • MBPcl3 and the anti-antibody were incubated either at different times or simultaneously in Microlon ELISA microplates with MBP previously fixed.
  • Figure 9 shows that the presence of MBPcl3 blocks binding of the antibody and the effect was stronger when MBPcl3 and anti-MBP were incubated together .
  • Nucleic acids are highly susceptible to the activity of endo- and exonucleases . Consequently, aptamers that are intended to be used as therapeutic agents must be developed to withstand the enzymatic activity of these nucleases.
  • the nucleotide modifications by phosphorothioates (PS), are well studied; however, the modification of an aptamer could trigger a change in activity so an aptamer MBPcl3 modified with nucleotide PS was tested (Figure 8A) .
  • Figure 8A shows that the modification of MBPcl3 by PS not only retained its activity, but also enhanced it.
  • the PS of MBPcl3 version was also capable of binding to myelin crude extracts (Figure 8B) .
  • SELEX is a proven methodology to obtain active nucleic acids with the ability to bind specific targets (for reviews, see 14, 15) . Essentially, SELEX consists in repeated binding, selection and amplification tests of aptamers from an initial library until enrichment of specific molecules is attained.
  • MS is an autoimmune, inflammatory and demyelinating disease that attacks the central nervous system; delaying, stopping or reverse the progression of the disease is yet to be achieved in patients with MS.
  • the MBP is one of the main components of the myelin sheath (20) and is found throughout the myelin sheath (21) .
  • Previous studies confirm that many MS patients are carriers of MBP autoantibodies and suggest that these antibodies may be responsible for the loss of myelin in the neuronal axons (1, 22-24) . Therefore, having MBP as a target protein could result in an immunoprotective therapy blocking the interaction between the autoantibodies and endogenous MBP protein.
  • the present invention provides specific aptamers against pure MBP from mice mainly containing 18.5 Kda-isoform in order to test them in a MS animal model.
  • Selected clones MBPcl3 and MBPcl9 showed a shorter randomized region exhibiting 26 nucleotides, thus enabling the binding activity of the MBP onto nitrocellulose membranes ( Figure 1A, B and 2A) and in the ELISA plates ( Figure 3) . It is noteworthy that it was found that the unselected aptamer library binds to the MBP applied to nitrocellulose membranes ( Figure IB) but not in the ELISA plates ( Figure 6A) . Indeed, it was demonstrated that MBP binding activity of MBPcl3 is sequence-specific.
  • MBPcl3 with its scrambled nucleotide sequence could not bind the MBP as good as MBPcl3 ( Figure 3) .
  • the secondary structure of aptamers MBPcl9 and MBPcl3 was predicted with mFold (12) in order to rationally identify potential active domains for both aptamers. Therefore, shorter versions of aptamers were synthesized; however, none of them was able to match the activity of MBPcl3 of total length ( Figure 4C and 5) .
  • MBPcl3 activity was also compared with a recently published aptamer selected against myelin extracts, called 3064 (13) .
  • aptamer 3064 demonstrated increased MBP binding activity; however, in all our studies it was not possible to reduce the standard deviation, which was very high. In contrast, when MBPcl3 and 3064 were tested with a complex protein mixture, MBPcl3 performed better. While MBPcl3 binding was related to the amount of MBP in the protein mixture, it was not so with 3064 ( Figure 6B) , suggesting that 3064 binds to non-specific molecules .
  • MBPcl3 Since MBPcl3 was obtained to be used as a useful therapeutic molecule for multiple sclerosis, its activity (of MBPcl3) was also compared with a commercial anti-MBP polyclonal antibody. When MBPcl3 and antibody were incubated with pure MBP, MBPcl3 could demonstrate increased MBP binding activity (Figure 7A) . MBPcl3 performance was also superior to the antibody when both were assessed for detecting the MBP in a complex protein mixture ( Figure 7B) . Furthermore, with the amount used for both molecules, the anti-MBP antibody could not resolve the presence of MBP in the protein mixture. When MBPcl3 and anti- MBP antibody were subject to competition for MBP, MBPcl3 could displace the binding of the antibody and the effect was greater when both molecules were incubated together ( Figure 7C) .
  • aptamers that are intended to be used as a therapeutic agent must overcome the activity of endo- and exonucleases circulating in the bloodstream.
  • a common solution to this problem is the use of aptamers with modified nucleotides.
  • the modification of an aptamer post-SELEX can alter its activity.
  • MBPcl3 was synthesized with modifications via phosphorothioate (PS) and its activity was measured. In fact, the modifications by PS did not alter MBPcl3 activity but, on the contrary, they enhanced it ( Figure 8A) .
  • MBPcl3 and its phosphorothioate analog also attained the binding to myelin crude extracts ( Figure 8B) .
  • the aptamer MBPcl3 and its phosphorothioate analog happen to be potential agents for diagnostic studies, where there is a need to detect the presence of MBP. Both molecules are also suitable as agents for the treatment of autoimmune diseases where MBP is involved. It is suggested that MBPcl3 can block the binding of autoantibodies to MBP, so that the recruitment of inflammatory cells and their degradation is blocked .

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Abstract

L'invention concerne des aptamères dirigés contre la protéine basique de la myéline (MBP), ainsi que leur utilisation comme agents neuroprotecteurs chez des patients atteints de sclérose en plaques et comme agents de détection de la MBP.
PCT/IB2014/066107 2013-11-21 2014-11-17 Aptamères utilisés comme agents neuroprotecteurs contre la protéine basique de la myéline Ceased WO2015075622A1 (fr)

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