EP3775212A1 - Identifizierung von muskulären mirnas als molekulare biomarker und co-adjuvans zur behandlung von spinaler muskelatrophie - Google Patents
Identifizierung von muskulären mirnas als molekulare biomarker und co-adjuvans zur behandlung von spinaler muskelatrophieInfo
- Publication number
- EP3775212A1 EP3775212A1 EP19722955.2A EP19722955A EP3775212A1 EP 3775212 A1 EP3775212 A1 EP 3775212A1 EP 19722955 A EP19722955 A EP 19722955A EP 3775212 A1 EP3775212 A1 EP 3775212A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- hsa
- inhibitor
- microrna
- sma
- subject
- 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.)
- Pending
Links
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Classifications
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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
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
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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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
- C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
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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
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/31—Combination therapy
Definitions
- the present invention relates to specific inhibitors of human microRNAs, for use in the treatment of spinal muscular atrophy (SMA) and pharmaceutical compositions including them.
- SMA spinal muscular atrophy
- the present invention further relates to the identification of markers with high sensitivity and specificity for SMA prognosis, prognostic methods including the use of such markers and prognostic kits for detecting such markers.
- SMA Spinal muscular atrophy
- SMA l-lll 3 infantile forms
- the gene responsible for all forms of SMA is localized in 5q13 and it is called SMN1 (Survival of Motor Neuron 1).
- the SMN1 gene is absent in homozygosis in 95-97% of SMA patients.
- SMN1 hypermorphic gene of SMN1, SMN2.
- the two genes are highly homologous and codify for the same protein (SMN), but due to the alternative splicing of exon 7, the genes SMN2 produce mainly a protein isoform deprived of this exon which is quickly degraded.
- SMA is characterized by progressive muscle paralysis due to the degeneration of the alpha-motor neurons of spinal cord.
- the role of the skeletal muscle in the condition physiopathology is still controversial, although there are several experimental pieces of evidence supporting an active pathogenetic role (Braun et al., 1995; Guettier-Sigrist et al. , 2002; Chan et al., 2003; Arnold et al., 2004; Kariya et al., 2008; Walker et al., 2008; Bricceno et al., 2014).
- SMA I or Werdning-Hoffmann disease
- SMA II or Dubowitz disease
- SMA III the motion acquisitions take place normally, however the patients can lose deambulation at variable age (D’Amico et al. , 2011).
- SMN2 is the currently known main modifier of phenotypical gravity
- the first identified biomarker was the determination of the number of copies of SMN2 (Feldkotter et al., 2002; Tiziano et al., 2007; Crawford et al., 2012): the identification of two copies of SMN2 is strongly predictive of a serious phenotype, as there is 80% of probability of a SMA I.
- the predictive power of the identification of 3 copies of SMN2 is considerably more limited, since they are observed in about 60% of SMA II and in 50% of SMA III.
- the finding of 4 copies of SMN2 is predictive of a lighter phenotype (Crawford et al., 2012).
- the object of the present invention is to provide prognostic biomarkers of spinal muscular atrophy (SMA) and new compounds useful in the prevention and/or treatment of this disease.
- SMA spinal muscular atrophy
- the present invention is based upon the scientific experiments shown in the examples.
- the authors have identified deregulated miRNAs in the patients affected by SMA, in particular they have identified three miRNAs (HSA-miR181a-5p, HSA-miR324- 5p and HSA-miR451a) over-expressed in the patients affected by SMA showing a correlation with the condition gravity.
- miR181 microRNA inhibitors in particular HSA-miR181a-5p inhibitors determine an increase in the survival of the mice affected by SMA.
- the present invention firstly relates to an inhibitor of the miR181 microRNA, in particular of HSA-miR181a-5p microRNA, for use in the prevention and/or treatment of spinal muscular atrophy (SMA), wherein said HSA-miR181a-5p microRNA has SEQ ID NO 1 and access number miRbase MIMAT0000256.
- SMA spinal muscular atrophy
- the present invention further relates to a pharmaceutical composition
- a pharmaceutical composition comprising as pharmacologically active agent an inhibitor of HSA-miR181a-5p microRNA and at least a pharmaceutically acceptable carrier.
- the present invention further relates to a Kit for the simultaneous, separate or sequential administration of an inhibitor of HSA-miR181 a-5p miR181 microRNA and one or more of the following compounds Nusinersen, HSA-miR324-5p microRNA inhibitor, HSA-miR451 a microRNA inhibitor.
- the present invention further relates to an in vitro method for evaluating the prognosis of spinal muscular atrophy (SMA) in a subject, comprising:
- the present invention further relates to a method for monitoring the development of spinal muscular atrophy in a subject, comprising:
- HSA-miR181 a-5p, HSA-miR324-5p and/or HSA-miR451 a microRNAs in two or more biological samples of said subject, wherein the samples were obtained at spaced time points, and
- the present invention further relates to a method for monitoring the effectiveness of a treatment of spinal muscular atrophy in a subject, comprising: a. determining the level of HSA-miR181 a-5p, HSA-miR324-5p and/or HSA-miR451 a icroRNAs in a biological sample of said subject obtained before the start of treatment, in particular individual and/or added up levels;
- step (b) determining the level of microRNA in one or more biological samples of said subject obtained during or after treatment, in particular individual and/or added up levels, and c. comparing the level of individual microRNAs and/or their sum determined in the steps (a) e (b), and optionally between different samples in step (b).
- FIG. 1 Heatmap related to the analysis of miRnoma of muscle biopsies of patients and controls.
- the graph shows the differential expression of miRNAs deregulated in the muscle biopsies.
- the blue strips designate the down-regulated miRNAs, whereas the yellow ones relate to the up-regulated miRNAs.
- the heatmap shows that the analysed groups generate two distinct clusters (CTRL vs SMA).
- Figure 2 deregulated miRNAs in the serum of SMA patients, emerging from a first validation performed through relative qRT-PCR on 10 samples SMA and 10 controls (*a£0.05).
- FIG. 3 Correlation graphs between the expression levels of miR-181a-5p, 324- 5p and 451 a and type of SMA.
- the boxplot shows the number of molecules/mI serum detected for miR-181a-5p (a), miR-324-5p (b) and miR-451a (c) in correlation with the type of SMA (I, II, III) ( * P£0.05).
- FIG. 4 Predictive power of miR-181a-5p, miR-324-5p and miR-451a in discriminating SMA patients from controls.
- the graphs show (a) the ROC curves of each miRNA analysed individually; (b) the ROC curve relative to the sum of the 3 miRNAs.
- FIG. 5 Survival curves of the mice SMA-A7 treated with single intrathecal injection of anti-miR-181a-5p or anti-miR-324-5p.
- the affected mice were treated on the day after birth with 0.5 nM of antagomir, of negative control or they were not subjected to treatment. From the obtained data it emerges that anti-miR-181 a-5p induces a statistically significative increase in the survival of the affected mice.
- FIG. 6 Treatment effect with Mimic of mir-181a-5p and miR-324-5p in cells of human neuroblastoma SH-SY5Y. Both miRs are almost unmeasurable under basal conditions (scramble). The transient transfection of Mimics determines a marked increase in the levels of single miRs. However, the levels of transcripts of the SMN1 and SMN2 genes remain unchanged. Only in case of miR-324-5p a decrease in the levels of isoform SMN-del7 is noted, the biological meaning remains unknown.
- MicroRNA are an endogenous sub-class of not codifying small RNAs constituted by single-filament ribonucleic acids, usually with a length of 21-22 nucleotides, having the capability of binding specific sequences of messenger RNA by inhibiting the subsequent translation into protein. Their presence guarantees a fine mechanism for adjusting the protein expression both under normal or pathological conditions.
- Inhibitor of microRNA (designated in the present text even as anti miRNA) modified oligonucleotide, complementary to a specific miRNA; when miRNAs and anti-miRNAs bind, the endogenous function of the micro-RNA is abolished.
- SMA spinal muscular atrophy, neurodegenerative recessive autosomal disease
- SMN survival of motor neuron, essential protein for the survival and function of the spinal motor neurons
- NGS next generation sequencing, massive parallel sequencing of molecules of DNA.
- the present invention thus relates to inhibitors of miR181 microRNA, in particular of miR181 HSA-miR181a-5p microRNA for use in the prevention and/or treatment of spinal muscular atrophy (SMA), wherein said HSA-miR181a-5p microRNA has SEQ ID NO 1 and access number miRbase MIMAT0000256.
- the inhibitor could be for example an antagomir, an antisense oligonucleotide or an inhibitor of RNA.
- the molecules more commonly used as inhibitors of microRNAs, included in the object of the present invention, are represented by antisense oligonucleotides, also called antimiR, the sequence thereof is wholly or partially complementary to the sequence, (which normally includes the so-called seed sequence) of the target microRNA, and that is of the microRNA to be inhibited.
- the sequence will be complementary at least by 90, 91 , 92, 93, 95 or 100%; it is likely that there is the inhibitory effect with at least 90% of complementarity.
- any inhibitor of selected HSA- miR181a-5p microRNA is a specific inhibitor for HSA-miR181a-5p micro RNA having SEQ ID NO 1 and access number miRbase MIMAT0000256, and that is an inhibitor inhibiting, that is silencing, only said microRNA and which is not capable of inhibiting, that is silencing, other microRNAs expressed in the same organism.
- the invention inhibitor will be an optionally chemically modified oligonucleotide having a sequence complementary to SEQ ID NO 1.
- the antimiRs (and that is the oligonucleotides inhibiting microRNAs) can be modified chemically with the purpose of increasing the affinity thereof for the target microRNA (thus by increasing the annealing temperature of the inhibitor- microRNA hybrid), of increasing the half-life thereof and/or, for example, in vivo biodistribution.
- the inhibitors of HSA-miR181a-5p, HSA-miR324-5p and HSA-miR451a microRNAs are chemically modified oligonucleotides.
- the person skilled in the art could draw, starting from what is commonly known in the state of art, several antimiRs suitable to the implementation of the invention.
- the modifications commonly used in the state of art include, for example, modifications to the sugar of nucleotide, modifications to the nitrogen base, modifications to the nucleic bond.
- the assays described in the experimental portion are sufficient to evaluate the effectiveness of the inhibitors drawn based upon the above teachings and methods and programmes are available allowing to evaluate the specificity of the same; thereamong, for example, programmes are available commonly used by the person skilled in the art for evaluating the possible crosslinking and then the specificity of the antimiR, such as BLAST, Vmatch, and RNAhybrid.
- the programme CrossLink for example, can be used for evaluating the potential interactions between microRNAs or anti-microRNAs and their target sequences.
- suitable inhibitors of microRNAs the invention relates to, can be purchased from producers specialized for such services and inhibitors specific for said microRNAs are also available on the market.
- inhibitors commercially available by Exiqon are the following ones: mmu-miR-181 a-5p batch#620620; mmu-miR-451a batch#183631 ; mmu-miR-324-5p batch#190693).
- inhibitors are the oligonucleotides having sequence SEQ ID NO 4 (LNA miRNA inhibitor mmu-miR-181 a-5p- product sequence 5’- C*G*A*C*A*G*C*G*T*T*G*A*T*G*T -3’) , SEQ ID NO 5 (LNA miRNA inhibitor mmu- miR-451 a- product sequence 5’- G * T * A * A * T * G * G * T * A * A * C * G * G * T * T-3’), SEQ ID NO 6 (LNA miRNA inhibitor mmu-miR-324-5p product sequence 5’- A*T*G*C*C*C*T*A*G*G*G*G*A*T*G*C-3’).
- the inhibitor of the present invention can be used in the prevention and/or treatment of SMA (of type l-lll).
- SMA of type l-lll
- HSA-miR181a- 5p microRNA inhibitor having SEQ ID NO 1 and access number miRbase MIMAT0000256 is intended for use in the prevention, treatment, reversion, cure or decrease in the process of pathogenesis correlated with SMA.
- the therapeutic treatment according to the present invention can be implemented with a formulation suitable to any administration route, preferably systemic, still more preferably intrathecal route. Such administration could be performed by several doses or by treatment with one single dose.
- HSA-miR181a-5p microRNA inhibitor will be administered in a therapy combined with the medicament Nusinersen and/or with an inhibitor of HSA- miR324-5p microRNA and/or with an inhibitor of HSA-miR451 a micro RNA, wherein said HSA-miR324-5p microRNA has SEQ ID NO 2 and access number miRbase MIMAT0000761 and said HSA-miR-451a micro RNA has SEQ ID NO 3 and access number miRbase MIMAT0001631.
- kits for the simultaneous, separate or sequential administration of an inhibitor of HSA-miR181 a-5p microRNA and one or more of the following compounds Nusinersen, inhibitor of HSA-miR324-5p micro RNA, inhibitor of HSA-miR451a microRNA, is also described.
- Such kit will be advantageously used for use in the prevention and/or treatment of spinal muscular atrophy (SMA).
- terapéuticaally effective amount means the amount of active compound or pharmaceutical agent inducing the biological or medical response in a system of tissues, in an animal or in a human being including alleviation, prevention, treatment or delay of the onset or of the progression of the symptoms of the disease or the treated disorder.
- composition is meant relating to even a product comprising the specific ingredients in the specific amounts, as well as any product resulting, directly or indirectly, from combinations of the ingredients specified in the specified amounts.
- composition then further relates to a pharmaceutical composition comprising an inhibitor as above defined and at least a pharmaceutically acceptable carrier.
- an inhibitor according to the present invention as active ingredient is mixed with a pharmaceutical carrier according to conventional techniques of pharmaceutical preparation, which carrier can have a wide variety of forms according to the preparation form wished for the administration.
- composition will include even one or more of the following pharmacologically active agents: Nusinersen, HSA-miR324-5p microRNA inhibitor, HSA-miR451a microRNA inhibitor.
- the compounds and preparations of the invention could be formulated for administration by any mode which results convenient for medical or veterinary purposes.
- Tablets and capsules for oral administration could correspond to one single dose, and could include conventional excipients such as, for example, binding agents, for example maize or glucose syrup, acacia or Arabic rubber, tragacanth rubber, gelatine, sorbitol, polyvinylpyrrolidone; filling agents, for example, lactose, sugar, maize starch, calcium phosphate, sorbitol or glycine; lubricants for tablets, for example magnesium stearate, talcum, polyethyleneglycol or silica; disintegrants, for example potato starch; and pharmaceutically usable humidifying agents, for example sodium lauryl sulphate.
- the tablets could be coated by following methods well known in the usual pharmaceutical practice.
- Liquid preparations for oral administration could assume, for example, the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or a dry product to be reconstituted with water or another suitable carrier before use.
- Such liquid preparations could include conventional additives, including, for example, stabilizing agents of the suspension, for example sorbitol, methyl cellulose, glucose syrup, gelatine, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate or hydrogenated edible fats: emulsifying agents, for example lecithin, sorbitan monooleate or acacia rubber: not aqueous carriers (which can include edible oils), for example almond oil, oily esters (for example glycerin), propylene glycol, or ethyl alcohol; preservatives, for example methyl- or propyl-hydroxybenzoate or sorbic acid; and, optionally, conventional colouring and flavouring agents.
- stabilizing agents of the suspension for example sorbitol, methyl cellulose, glucose syrup, gelatine, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate or hydrogenated edible fats: emulsifying agents, for example lecithin, sorb
- compositions according to the present invention could suitably include an inhibitor as herein described or a derived or solvated salt thereof from 0.03% to 95% by weight with respect to the composition.
- each dose could include for example 0.1 to 1000 mg of compound.
- the invention also provides a method for use in the prevention and/or treatment of spinal muscular atrophy (SMA) comprising a step of administration to a human subject requiring it, a therapeutically effective amount of HSA-miR181a-5p microRNA inhibitor having SEQ ID NO 1 and access number miRbase MIMAT0000256 as defined in the present description and in the claims optionally in association to the other herein described active agents.
- SMA spinal muscular atrophy
- the invention also provides the following methods:
- a method for evaluating the prognosis of spinal muscular atrophy (SMA) in a subject comprising:
- a method for monitoring the development of spinal muscular atrophy in a subject comprising:
- HSA-miR181 a-5p, HSA-miR324-5p and/or HSA-miR451 a microRNAs in two or more biological samples of said subject, wherein the samples were obtained at spaced time points, and
- a method for monitoring the effectiveness of a treatment of spinal muscular atrophy in a subject comprising:
- a determining the levels of HSA-miR181 a-5p, HSA-miR324-5p and/or HSA-miR451 a microRNAs in a biological sample of said subject obtained before the start of treatment; b. determining the levels of HSA-miR181 a-5p, HSA-miR324-5p and/or HSA-miR451 a microRNAs in one or more biological samples of said subject obtained during or after treatment, and
- step (b) comparing the levels of HSA-miR181 a-5p, HSA-miR324-5p and HSA-miR451a microRNAs determined in steps (a) and (b), and optionally between different samples in step (b), for example 1 , 3, 6, 12, 24, 36 or 48 months earlier.
- biological samples useful in anyone of the methods of the invention include plasma, serum, urine, muscle biopsies, skin biopsies, cerebrospinal liquid.
- microRNAs are extracted and purified from any biological sample.
- anyone of the above-described methods can further include the step of reducing or eliminating the degradation of microRNAs.
- control level as used herein, comprises predetermined standards (for example, a value published in a reference) and levels determined experimentally in samples analysed and processed by control subjects (for example healthy subjects of equal age, patients treated with placebo, etc.).
- a Kit is also herein described for use in the prognosis of spinal muscular atrophy in a subject comprising reagents for the determination of levels of HSA-miR324-5p micro RNA and/or of HSA-miR451a micro RNA in a biological sample and optionally one or more control samples, that is samples wherein the levels of microRNAs are known.
- the reagents for the measurement of the levels for determining the levels of the microRNAs are known to the person skilled in the art and exemplified in the examples.
- the high-throughput analysis of miRnoma of the patients affected by SMA was performed on 7 muscle biopsies (3 SMA I, 2 SMA II and 2 SMA III), and 7 controls obtained by the Division of Child Neuropsichiatry of the Neurological Institute“Carlo Besta” in Milan.
- the samples were selected based upon the biopsy site selected (quadriceps femoris) and the execution of sampling at the first stages of the disease to avoid the excessive presence of fibrotic tissue.
- the controls were selected among morphologically normal samples, obtained from healthy subjects of the same range of age.
- the total RNA was extracted from muscle biopsies by the use of TRIzol® Reagent (Life Techologies) in a ratio of 1 ml/100 mg of sample, according to the producer’s protocol.
- the extracted RNA was re-suspended in water RNase-free and quantified by reading at spectrophotometer NanoDrop 1000 (Thermo Scientific).
- the total RNA was at first quantified and qualified by Byoanalyzer (Agilent) with the purpose of evaluating quality and integrity thereof, defined through the number of integrities of RNA (RIN).
- the fraction of small RNAs, containing the miRNAs, was selected through cutting from not-denaturing 6% polyacrylamide gel.
- the extraction of miRNAs from serum was performed by using miRCURYTM RNA Isolation Kit - Biofluids (EXIQON). With the purpose of avoiding the presence of blood cells in the serum, the sample was centrifuged at 3.000rpm for 5 minutes before proceeding with extraction. The miRNAs were extracted from an initial volume of serum of 200mI for each sample. In the final extraction step, a digestion with DNase RNase-free for 15 minutes at room temperature was performed in order to remove the residues of cell-free DNA.
- the retrotranscription of miRNAs extracted from serum was performed with Universal cDNA synthesis kit II (EXIQON), according to the producer’s instructions.
- the reaction of retrotranscription was performed under different conditions.
- 4mI of RNA were retrotranscripted thereto 4mI 5x Reaction Buffer, 2mI Enzyme mix, 1 mI Synthetic RNA spike in (UniSp6) and 9mI H20 nuclease-free, in a final volume of 20mI, were added.
- 2mI Enzyme mix 1 mI Synthetic RNA spike in (UniSp6)
- 9mI H20 nuclease-free in a final volume of 20mI
- miRNA quantification in serum through absolute qRT-PCR assoluta 2mI of RNA were retrotranscripted thereto 2mI 5x Reaction Buffer, 1 mI Enzyme mix and 5mI H20 nuclease-free, in a final volume of 10mI, were added.
- the obtained cDNA was brought to a 100x concentration of the final dilution with H20 nuclease-free for the subsequent reactions of relative qRT-PCR, or at a 25x concentration of the final dilution in TE pH8 (10mM Tris-HCL, 1 mM EDTA) for the subsequent reactions of absolute qRT-PCR.
- the libraries were sequenced on Genome Analyze I lx (lllumina), according to the producer’s directions.
- the obtained reads (having length of 31 bases, in single-end) were at first filtrated by quality, by using FASTX-toolkit, and then subjected to trimming (by means of Trim galore) for removing the adapter. Only the reads longer than 15 bases were preserved for the subsequent analysis.
- the filtered reads were aligned to the sequences of the precursor miRNAs noted in the miRBase (www.mirbase.org) database. For the alignment Bowtie algorithm was used, by allowing a maximum of two mismatches.
- the identification and quantification of mature miRNAs were performed by using exclusively the reads perfectly aligned to the sequence of each mature miRNA.
- the reads not aligned in miRBase were subsequently filtered, by removing potential molecules of tRNA, rRNA or other products noted in Rfam, ncRNA and NONCODE databases.
- the remaining reads were grouped together with the identification of possible miRNAs still not noted, performed by miRDeep (v2).
- miRDeep the evaluation of the differential expression of the miRNAs identified between SMA samples and controls was performed through the software edgeR (v2.4.1).
- the so-identified miRNAs were considered expressed in differential way between patients and controls exclusively for values of FDR (False Discovery Rate) ⁇ 0.05.
- primer reverse included the sequence of the universal primer used in the commercial kit Universal cDNA synthesis kit II (EXIQON), followed by a queue of T15 and by 4 nucleotides complementary to the last 4 of the end 3’ of the sequence of the mature miRNA.
- primer forward had a variable length and included the sequence of mature miRNA (www.mirbase.org), followed by a queue of A15 and by 3 nucleotides complementary to the last 3 of the end 3’ of the universal primer R.
- PCR In order to transform into double-filament DNA the fragment containing the sequence of mature miRNA, one single cycle of PCR was performed.
- the reaction of PCR was performed in a final volume of 12.5mI including: 0.1 mI GC-Platinum Taq DNA Polymerase (5u/mI), 1.25mI 10x Reaction Buffer, 1 mI 25mM MgCI2, 0.25mI 10mM dNTPs, 1 mI primer exst_F 10mM, 1 mI primer exst_R 10mM e 7.9mI H20 nuclease-free.
- the cycle of PCR was: 30” - 94°C, 30” - 54°C, 30” - 72°C.
- the product obtained from the previous reaction was cloned in the vector pDrive (Qiagen).
- the pDrive was at first linearized with EcoRV (Roche) by incubating at 37°C for 1 hour 10pg of the vector with 10 U of enzyme, 2.5mI 10x SuRE/Cut Buffer B and 2dH20 in a final volume of 25mI.
- the digested vector was subsequently purified by precipitation with sodium acetate 3M (C2H3Na02) pH 5.2.
- the digested vector was made T-overhang by amplification in presence of only dTTPs, with the purpose of favouring the pairing thereof with the A- overhang existing in the inserts.
- Such reaction was performed in a final volume of 12.5mI with 0.2mI GC-Platinum Taq DNA Polymerase (5u/pl), 1.25mI 10x Reaction Buffer, 0.75mI 25mM MgCI2, 0.25mI 10mM dTTPs, 300ng of the linearized vector pDrive and 2dH20 at volume. The reaction was incubated for 20 minutes at 72°C.
- the ligation was performed in a final volume of 20mI with 5mI 4x AnzaTM T4 DNA Ligase Master Mix (Invitrogen), 1 mI vector pDrive EcoRV T-overhang [50ng/pl], 10ng of the insert. The reaction was incubated at room temperature for 30 minutes.
- Competent bacteria EZ were transformed with the vector pDrive including the fragment of interest according to the method of the heat shock: 30 minutes in ice, 50 seconds at 42°C and 2 minutes in ice. Then 250mI of SOC medium (2% tryptone, 0.5% 10mM NaCI, 2.5mM KCI, 10mM MgCI2, 10mM MgS04, 20mM Glu) were added; the bacteria were incubated for 1.5 hours at 37°C under stirring at 300 rpm. Subsequently, they were plated on petri including LB-AGAR with ampicillin 100pg/ml, X-Gal 80pg/ml, IPTG 50mM and incubated O/N at 37°C.
- SOC medium 2% tryptone, 0.5% 10mM NaCI, 2.5mM KCI, 10mM MgCI2, 10mM MgS04, 20mM Glu
- the cycle of PCR was: 5’ - 95°C; 1’ - 95°C, 1’ - 56°C, 1’ - 72°C (for 30 cycles); 5’ - 72°C.
- the size of the obtained amplified ones was evaluated by electrophoresis on agarose gel: only the colonies producing products of PCR> of -200 bp were sequenced.
- the sequence of each cloned miRNA was checked by means of sequencing of Sanger.
- the product of PCR (5mI) was purified with 1 mI ExoSAP-IT® PCR Product Cleanup (Affymetrix) for 15 minutes at 37°C, thereafter ExoSAP was inactivated at 80°C for 15 minutes.
- the fragment of DNA of interest was sequenced by using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), according to the producer’s directions, in a final volume of 10mI.
- the sequence reactions were purified with BigDye® XTerminatorTM Purification Kit (Applied Biosystems), according to the producer’s protocol.
- the sequencing was performed by capillary electrophoresis on automatic sequencer ABI-Prism 3130 (Applied Biosystem).
- the plasmid DNA was extracted with E.Z.N.A Plasmid DNA Mini Kit I Spin Protocol (OMEGA Biotek) according to the producer’s protocol.
- the concentration of the extracted DNA was quantified by spectrophotometer MultiskanTM Go (Thermo Scientific) and its integrity was verified through electrophoretic run on agarose gel. The correctness of the insert sequence was again verified.
- the extracted plasmid DNA was diluted at a final concentration of 3ng/pl by means of serial dilutions for the construction of the standard curves.
- each antagomir was diluted 1 : 1 in Cerebro Spinal Fluid (CSF) artificial 2x (238 mM NaCI, 52.4 mM NaHC03, 5 mM KCI, 2 mM NaH2P04, 2.6 mM MgCI2, 20 mM glucose).
- CSF Cerebro Spinal Fluid
- the injection at intra-cerebro-ventricular P2 of the affected mice with 0.5 nM of each antagomir (in a final volume of 1 mI) was performed. The phenotypical effect of the treatment was evaluated exclusively through survival.
- the AQ (Absolute Quantification) files obtained from relative qRT-PCRs were converted into RQ (Relative Quantification) files by means of RQ Manager 1.2 and subsequently analysed with Real Time StatMiner® v4.1. For the identification and subsequent removal from the analysis of the outliers the Grubbs’ test was applied. Spike-In UniSp6 was used as normalizer in the relative qRT-PCRs. The comparison of the levels of different miRNAs between patients and controls was performed by using the not-parametric test Wilcoxon and only the miRNAs with FDR (False Discovery Rate) ⁇ 0.05 were considered significant. The statistical analysis of the data obtained from absolute qRT-PCR was performed by the software Statgraphics Centurion XV (StatPoint Inc.).
- sensitivity and specificity of the determination of the levels of miRNAs in the patients with respect to the controls were determined by means of ROC (Receiver Operator Characteristic) curves.
- ROC Receiveiver Operator Characteristic
- the object of the present invention can be synthetized in two fundamental points: 1) the identification of 3 miRNA produced by the skeletal muscle, deregulated in the SMA patients, as biomarkers for the condition.
- a method of absolute qPCR was developed allowing to determine the levels of miRNAs in serum (expressed as number of molecules/mI of serum); 2) the therapeutic application of the modulation of such miRNA under neurodegenerative conditions such as, apart from SMA, amyotrophic lateral sclerosis (SLA).
- SLA amyotrophic lateral sclerosis
- miRNAs some play a key role in the muscle physiology; some of them were previously identified as biomarkers for other neuro-muscle conditions (miR-1 , miR-133a/b and miR-206). Moreover, our data show that the miRNAs involved in processes of atrophy/denervation are deregulated even in SMA and their modulation could be a valid therapeutic target to slow-down the disease progression.
- Step 1 we tested the expression levels of the 99 deregulated miRNAs emerged from the analysis of the muscle biopsies on samples of serum of 10 SMA patients and 10 controls by means of relative qRT-PCR with commercial assays (Exiqon, miRCURY LNA). Commercial tests were available for 76/99 miR deregulated in the muscle biopsies.
- the relative quantification was performed by using a spike-in, Unisp6, as calibrator.
- Step 2 For a value of alfa £0.05, 21 miRNAs were significantly over-expressed and 3 under-expressed in serum of the SMA patients with respect to the controls, thus resulting potential biomarkers (Figure 2)
- Step 2 For the 24 miRs detected in Step 1 and for the 23 miRs therefor commercial assays were not available, we developed an in-house system for absolute qRT-PCR. By using such assays, we analysed the expression levels of 47/99 miRs identified from the analysis of miRnoma on 91 samples of serum (51 of patients affected by different forms of SMA and 40 of control healthy subjects, Figure 2).
- miRNAs Most part of analysed miRNAs could not be dosed or were not expressed differentially between patients and controls; 3 miRNAs (miR324-5p, miR181a-5p e miR-451a) resulted to be promising biomarkers for SMA, as significantly over-expressed in samples of serum of the patients. Particularly interesting, some studies demonstrated the involvement of miR-181 a-5p and of miR- 451 a in controlling the myogenic differentiation processes (Naguibneva et al. , 2006 and Dmitriev et al., 2013). Afterwards, we evaluated the applicability of the above-mentioned miRNAs as biomarkers for SMA, by analysing possible correlations between the serum levels and some clinical parameters.
- the predictive power of the quantification of (single or combined) miRNAs in distinguishing the patients from the controls was evaluated. To this purpose, we constructed ROC curves.
- miRNA Most predictive miRNA is miR- 181 a-5p: by fixing the cut-off at 70.5 molecules/mI, the determination of miRNA has a diagnostic sensitivity of 75% and a specificity of 61 % (Figure 4a); the sum of the 3 miRNAs increases sensitivity and specificity: in particular, by fixing the cut-off at 380 molecules/mI, sensitivity and specificity reach 80 and 74%, respectively ( Figure 4b).
- Figure 4a the sum of the 3 miRNAs increases sensitivity and specificity: in particular, by fixing the cut-off at 380 molecules/mI, sensitivity and specificity reach 80 and 74%, respectively.
- each antagomir into 5 mice SMNA7 and a control antagomir- scramble into other 5.
- P2 postnatal life
- the inventors have identified deregulated miRNAs in SMA patients with respect to the controls.
- the primary objective of the study was to identify the biomarkers for the condition: 1) the modulation thereof was independent from the products of the SMN2 genes and 2) which derived from a tissue (the skeletal muscle) which has a central role in the pathogenetic mechanism of the condition.
- the skeletal muscle either it is active, as retrograde signal on the spinal motor neuron, or passive, after denervation and triggering of the atrophy processes
- the rational behind the selection of this approach was to identify deregulated miRNAs on tissues deriving from patients.
- the deregulated miRNAs in the skeletal muscle were identified by analysis of miRNoma of muscle biopsies and they were subsequently quantified in more than 90 samples of serum of patients and controls. Out of 99 deregulated miRNAs in the skeletal muscle of the patients, only 3 could be dosed and/or deregulated in serum of the patients. It is to be noted that typical myomiRs, such as miR-1 , miR-133 and miR- 2016, over-expressed in the serum of patients affected by DMD (Cacchiarelli et ai, 2011) or miR usually released into circulation during processes of cellular death, are not deregulated in SMA sera.
- HSA-miR181 a-5p, HSA-miR324-5p and HSA-miR451 a are over-expressed in the patients and allow to differentiate patients and controls with a sensitivity of 80% and a specificity of 74% (p ⁇ 0.0001).
- HSA-miR324-5p and HSA- miR451a are also correlated with the phenotypical gravity, since they have significantly higher levels in the less serious forms of SMA.
- HSA-miR181a-5p can be considered a therapeutic approach for SMA, in combination with other treatments, but it can be effective even in other neurodegenerative diseases such as SLA, which has several features in common with SMA. It would be further interesting to evaluate the application thereof under other neurodegenerative conditions of SNC, such as Alzheimer and Parkinson disease.
- the analysed samples are particularly precious and rare as deriving from muscle biopsies performed with diagnostic purpose several years ago: it has to be taken into account that, considering the bioavailability of a highly sensitive and specific genetic test, the muscle biopsy is a by now obsolete procedure in the diagnostic process of SMA, apart from being ethically not acceptable.
- the miRNAs deregulated in the serum of the patients with respect to the controls could be identified directly.
- This approach differently from the published studies not only in the field of SMA but even of other conditions, allowed to detect 3 deregulated miRNAs, but above all to give a therapeutic apart from prognostic/pathogenic meaning to the invention.
- the finding of greatest translational impact of the study is the definition of the therapeutic role of modulation of HSA-miR-181 a-5p.
- SEQ ID NO 1 AACAUUCAACGCUGUCGGUGAGU HSA-miR181 a-5p micro RNA has SEQ ID NO 1 and access number miRbase MIMAT0000256;
- SEQ ID NO 3 AAACCG U U ACC AU U ACUG AG U U HSA-miR-451 a micro RNA has SEQ ID NO 3 and access number miRbase MIMAT0001631 ; SEQ ID NO 4 (LNA miRNA inhibitor mmu-miR-181 a-5p product sequence 5’-
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PCT/IB2019/052820 WO2019197954A1 (en) | 2018-04-10 | 2019-04-05 | IDENTIFICATION OF MUSCULAR miRNAS AS MOLECULAR BIOMARKERS AND CO-ADJUVANT FOR THE TREATMENT OF SPINAL MUSCULAR ATROPHY |
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