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WO2024175682A1 - Procédé de préparation d'une préparation d'arn et son utilisation - Google Patents

Procédé de préparation d'une préparation d'arn et son utilisation Download PDF

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Publication number
WO2024175682A1
WO2024175682A1 PCT/EP2024/054459 EP2024054459W WO2024175682A1 WO 2024175682 A1 WO2024175682 A1 WO 2024175682A1 EP 2024054459 W EP2024054459 W EP 2024054459W WO 2024175682 A1 WO2024175682 A1 WO 2024175682A1
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Prior art keywords
rna
sample
blood
cells
subject
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English (en)
Inventor
Peter Bauer
Christian Beetz
Ruslan AL-ALI
Mandy RADEFELDT
Sabrina LEMKE
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Centogene GmbH
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Centogene GmbH
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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention is related to a method for preparing an RNA preparation, a method of sequencing an RNA molecule in a sample using such RNA preparation, a method for quantifying an RNA molecule in a sample using such RNA preparation, a method for determining the course of a disease in a subject using such RNA preparation, a method for monitoring an effect of a therapy administered to a subject suffering from a disease using such RNA preparation, a method for determining whether a subject suffering from a disease is likely to respond to a treatment using such RNA preparation, a method for determining the presence or absence of a splice variant of a gene in a sample using such RNA preparation, a method for determining a relative abundance of an individual RNA molecule in a sample using such RNA preparation, a method for determining allele-specific expression of a gene in a plurality of samples, and a method for determining the proportion of subjects of a group of subjects carrying an allele of a gene using such RNA preparation.
  • RNA molecules are essential for interpreting the functional elements of the genome and understanding development and disease.
  • RNA transcriptome initially encompassed only protein-coding mRNA transcripts. Nevertheless, several RNA subtypes with distinct functions exist. Many RNA transcripts do not code for protein or have different regulatory functions in the process of gene transcription and translation. RNA types which do not fall within the scope of the central dogma of molecular biology are non-coding RNAs which can be divided into two groups of long non-coding RNA and short non-coding RNA.
  • Long non-coding RNA includes all non-coding RNA transcripts that are more than 200 nucleotides long. Members of this group comprise the largest fraction of the non-coding transcriptome.
  • Short non-coding RNA includes the following members: transfer RNA (tRNA), micro RNA (miRNA), Micro RNAs, small interfering RNA (siRNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA) and enhancer RNA (eRNA).
  • RNA sequencing also called massively parallel sequencing and NGS which is a high-throughput method used to determine a portion of the nucleotide sequence of an individual’s genome
  • RNA sequencing abbreviations: RNA sequencing (abbr. RNA-Seq)
  • RNA-Seq RNA sequencing
  • RNA-Seq provides a more detailed and quantitative view of gene expression, alternative splicing, and allele-specific expression.
  • RNA Integrity Number [RIN] > 7 RNA Integrity Number
  • DBS dried blood spot
  • DBS are widely used for diagnosis, in particular for the diagnosis for hereditary diseases
  • DBS in diagnostics is mainly limited to DNA sequencing (genetic testing), biomarkers (if the biomarker is to be analyzed is stable) and enzymatic analysis (if the enzyme to be analyzed is stable).
  • DNA sequencing genetic testing
  • biomarkers if the biomarker is to be analyzed is stable
  • enzymatic analysis if the enzyme to be analyzed is stable.
  • Only few working samples show the use of RNA-seq from DBS for research analysis, due to the low stability of the RNA and the limited amount of RNA, in particular mRNA, obtained from DBS, which can affect the accuracy and sensitivity of the RNA-seq analysis.
  • the problem underlying the present invention is the provision of a method for preparing an RNA preparation that leads to sufficient amounts of RNA, in particular mRNA, with sufficient quality that allows its use for RNA-Seq, gene expression analysis and diagnosis of diseases, in particular rare diseases.
  • a further problem underlying the present invention is the provision of an RNA preparation which is suitable for various applications and further processing steps, whereby such RNA preparation is obtained from a blood sample which can be easily stored and shows no relevant change in its composition, at least no relevant change which interferes with the RNA preparation’s application and further processing steps to which the RNA preparation is subjected.
  • Such application encompasses the use in the field of biotechnology, biochemistry, medicine, therapy and diagnosis.
  • a method for preparing an RNA preparation comprising a target RNA comprising a target RNA
  • the method comprises a step of extracting an RNA from a first sample, wherein the first sample is a dried blood sample, and wherein the step of extracting provides a second sample comprising the extracted RNA, a step of depleting a non-target RNA from the second sample, wherein the non-target RNA is selected from the group comprising a hemoglobin RNA, rRNA and a combination of a hemoglobin RNA and rRNA, wherein the step of depleting provides a third sample comprising the target RNA and from which the non-target RNA is depleted, and optionally a step of enriching the target RNA in or from the third sample, wherein the target RNA is enriched by capturing the target RNA by poly(A) tail capturing and wherein the step of enriching provides a fourth sample comprising the enriched target RNA, wherein the step of extracting an RNA from the first sample, wherein
  • a method for preparing an RNA preparation comprising a target RNA comprises a step of extracting an RNA from a first sample, wherein the first sample is a dried blood sample, and wherein the step of extracting provides a second sample comprising the extracted RNA, a step of enriching the target RNA in or from the second sample, wherein the target RNA is enriched by capturing the target RNA by poly(A) tail capturing and wherein the step of enriching provides a third sample comprising the enriched target RNA, and optionally a step of depleting a non-target RNA from the third sample, wherein the non-target RNA is selected from the group comprising a hemoglobin RNA, rRNA and a combination of a hemoglobin RNA and rRNA, wherein the step of depleting provides a fourth sample comprising the target RNA and from which the non-target RNA is depleted, wherein the step of extracting an RNA from a first sample, wherein the first sample is a dried blood sample,
  • a method of sequencing an RNA molecule in a sample comprises determining the nucleotide sequence of the RNA molecule and the sample is an RNA preparation prepared by a method as defined according to the first aspect, including any embodiment thereof.
  • a method for quantifying an RNA molecule in a sample comprises determining an amount of the RNA molecule in the sample, and wherein the sample is an RNA preparation prepared by a method according to the first aspect, including any embodiment thereof.
  • a method for determining the course of a disease in a subject comprises determining an amount of an RNA molecule in a sample from the subject at various points in time, wherein the RNA molecule is a prognostic marker for the disease and the sample is an RNA preparation prepared by a method as defined according to the first aspect, including any embodiment thereof.
  • a method for monitoring an effect of a therapy administered to a subject suffering from a disease comprising determining an amount of an RNA molecule in a sample from the subject after the therapy was administered to the subject and at several points in time thereafter, wherein the amount of the RNA molecule is indicative of the effect of the therapy and wherein the sample is an RNA preparation prepared by a method as defined according to the first aspect, including any embodiment thereof.
  • a method for determining whether a subject suffering from a disease is likely to respond to a treatment comprises detecting an RNA molecule in a sample from the subject, wherein the RNA molecule is a predictive marker for the disease and the sample is an RNA preparation prepared by a method as defined according to the first aspect, including any embodiment thereof.
  • a method for determining the presence or absence of a splice variant of a gene in a sample wherein the splice variant comprises a splice junction
  • the method comprises determining whether the splice junction is present and if the splice junction is present, the splice variant is present and if the splice junction is absent, the splice variant is absent
  • the sample is an RNA preparation prepared by a method as defined according to the first aspect, including any embodiment thereof.
  • a method for determining a relative abundance of an individual RNA molecule in a sample compared to the abundance of a plurality of RNA molecules in a sample comprises determining an amount of the individual RNA in a sample, wherein the sample is an RNA preparation prepared by a method as defined according to the first aspect, including any embodiment thereof, and determining the relative amount of the individual RNA in a sample to the amount of at least one RNA molecule of the plurality of RNA molecules in a sample.
  • a method for determining allele-specific expression of a gene in a plurality of samples wherein a plurality of alleles of the gene exist, wherein the method comprises determining the nucleotide sequence of an RNA transcript of the gene in each and any sample of the plurality of samples, allocating each nucleotide sequence to an allele of the plurality of alleles of the gene, and calculating based on such allocation the relative abundance of an allele relative to a different allele of the gene and/or to the plurality of alleles of the gene, and wherein the sample is an RNA preparation prepared by a method as defined according to the first aspect, including any embodiment thereof.
  • a method for determining the proportion of subjects of a group of subjects carrying an allele of a gene that also expresses an associated trait, wherein a plurality of alleles of the gene exist comprises determining the nucleotide sequence of an RNA transcript of the gene in a sample from each and any subject of the group of subjects, allocating each nucleotide sequence to an allele of the plurality of alleles of the gene, and calculating based on such allocation the relative proportion of subjects of the group of subjects that expresses the associated trait, and wherein the sample is an RNA preparation prepared by a method as defined according to the first aspect, including any embodiment thereof.
  • any method subject to the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh aspect of the present invention can form part of any method subject to the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh aspect of the present invention, including any embodiment thereof.
  • any method subject to the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh aspect of the present invention, including any embodiment thereof may, in an embodiment, encompass the method according to the first aspect and the method according to the second aspect, including any embodiment thereof, and thus the steps subject to the method according to the first aspect and the steps subject to the method according to second aspect, including any embodiment thereof.
  • RNA preparation which, starting from a blood sample, provides a sufficient amount of RNA, in particular mRNA, with sufficient quality which allows the use of such RNA preparation in RNA- Seq and, therefore, the use of such RNA preparation in any method where RNA is subject to, among others, sequencing or detection.
  • sample means preferably a limited quantity of a subject's material, wherein said subject's material is part of or has been taken from a subject and/or a subject's body.
  • said material is selected from the group comprising body fluids such as blood, a blood product, urine, saliva, cerebrospinal fluid and lymph, as well as stool or any kind of tissue and or cell material being part of a subject and/or a subject's body. More preferably, said material is a particulate compartment of blood comprising cells or a non-particulate compartment of blood or a combination of a particulate compartment of blood comprising cells and of a non-particulate compartment of blood.
  • said material is blood optionally comprising blood cells.
  • said material is blood cells. It will be acknowledged by a person skilled in the art that the presence of and/or an RNA in said sample is intended to be similar to and represent the presence and/or the level of the RNA in a larger amount of that subject's material. More precisely and as an illustrative, non-limiting example, a level of the RNA determined in a sample of, e.g., some ml of blood from a subject also represents a level of said RNA in the blood of the subject's body.
  • a sample from the subject comprises said subject's material in a form, for example processed, fixed and/or preserved such that said sample is suitable for use in the methods of each and any aspect of the invention.
  • the subject's material in the sample may thus be diluted, for example with a solvent suitable for the method of each and any aspect of the invention such as methanol and/or water, may be dried, for example on a filter card, may be resolved after having been dried such, for example with a solvent suitable for the method of the invention such as methanol and/or water, or a substance may be added, wherein said substance prevents blood from coagulation such as for example EDTA, heparin or citrate.
  • a solvent suitable for the method of each and any aspect of the invention such as methanol and/or water
  • RNA RNA
  • PAXGeneTM Blood Tubes RNA samples collected in a tube with EDTA are referred herein as EDTA blood sample.
  • a sample as preferably used in connection with each and any aspect of the present invention is prepared from a primary source such as whole blood.
  • Other samples include, but are not limited to, serum samples, plasma samples and blood cells.
  • whole blood is venous blood or capillary blood.
  • Venous blood is taken from the vein by taking a blood sample.
  • Capillary blood taken from the finger by taking a blood sample by use of a finger prick.
  • Blood contains many types of blood cells: white blood cells (such as monocytes, lymphocytes, neutrophils, eosinophils, basophils, and macrophages), red blood cells (erythrocytes), and platelets.
  • white blood cells such as monocytes, lymphocytes, neutrophils, eosinophils, basophils, and macrophages
  • red blood cells erythrocytes
  • platelets white blood cells [such as monocytes, lymphocytes, neutrophils, eosinophils, basophils, and macrophages]
  • red blood cells [erythrocytes]
  • platelets red blood cells [erythrocytes]
  • the sample is, in an embodiment, processed such that it is collected on a dry blood filter card (also referred herein as dried blood filter card or filter card).
  • a dry blood filter card also referred herein as dried blood filter card or filter card.
  • Such sample is also referred to herein either as a dry blood filter sample, dried blood filter sample, a dry blood spot on a filter card or a dried blood spot on a filter card.
  • Dried blood filter cards comprise an area consisting of filter paper where the blood can be dripped on, whereby the place where the blood should be dripped on is marked by a circle.
  • Dependent of blood volume dripped on the filter paper the spot can have a different size.
  • Such a spot can have circle area of 190 mm2, e.g. if 100 pl of EDTA blood was dripped on the filter paper.
  • a filter paper preferably 50 to 60 pl.
  • 50 pl to 60 pl corresponds to 3 drops of blood.
  • a person skilled in the art will acknowledge that the exact volume thus collected may vary depending on the hematocrit of the specific patient.
  • the filter paper of the filter cards is manufactured from 100% pure cotton fiber with no wet strength additives.
  • the critical physical properties during manufacturing are basis weight, ph and ash.
  • Basis weight should 110 lb +/- 5% per ream (179 g(m3 +/- 5%).
  • a ream is defined as 500 sheets 24” x 36” (ASTM D646-96).
  • the pH should ne 5.7 to 7.5 (test method ISO 6599:1981).
  • the ash should have a maximum of 0.1% (test method A o ASTM D586-97a).
  • Filter papers that meet the properties are Ahlstrom 226 filter paper/ specimen collection paper (also referred to as Ahlstrom Grade 226 filter paper) and Whatman® Grade 903 filter paper/ Whatman Body Fluid Collection Paper (Whatman BFC 18. K932661).
  • a specific design or type of a filter card is the CentoCard®. Beside parts that are needed for information and for ordering test, the CentoCard® comprises like other filter cards an area consisting of filter paper where the blood can be dripped on, whereby the place where the blood should be dripped on is marked by a circle.
  • the fdter paper of the CentoCard® is the Ahlstrom 226 filter paper/ specimen collection paper.
  • RNA Ribonucleic acid
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • 7SL RNA or SRP RNA signal recognition particle RNA
  • the RNA is extracted from blood.
  • blood comprises a cellular compartment, preferably comprising blood cells, and a non-cellular compartment typically comprising the parts and compartment, respectively, which is different from the cellular compartment of the blood.
  • the non-cellular compartment comprises serum and/or plasma.
  • RNA extracted from whole blood samples contains high levels of hemoglobin RNAs/ hemoglobin mRNAs (also referred to as globin RNAs/ globin mRNAs) from the dominant red blood cell component. These high abundance transcripts can interfere with sensitive measurement of the rest of the blood transcriptome.
  • the RNA preparation is a preparation comprising an mRNA, where the content, either the absolute content or any relative content, of the mRNA is increased compared to the content of the mRNA in the blood or blood cells from which the RNA is extracted.
  • the RNA preparation is an mRNA preparation, where the content, either absolute content or any relative content, of the mRNA is higher than the content of any other RNA contained in said mRNA preparation.
  • extracting an RNA from blood or blood cells means removing, separation and/or isolation RNA from other blood or blood cell components.
  • depleting an RNA from a sample that comprises RNA extracted from blood or blood cells means the reduction in the number or quantity of one or more species of RNA in the samples, preferably the reduction of the species hemoglobin RNA and rRNA.
  • lysing is a process of disintegration or dissolution (as of cells).
  • degradation, digestion or cleavage is the process of mechanically, chemically and/or enzymatically breaking molecules such as proteins or nucleic acids (such as RNA and DNA) into smaller fragments and/or components.
  • Enzymes digesting or cleaving proteins are known as proteinases.
  • Enzymes digesting or cleaving RNAs are known as RNAses.
  • Enzymes digesting or cleaving DNAs are known as DNAses.
  • Compounds that protect RNA from the activity of enzymes digesting or cleaving RNAs are known as RNAse inhibitors, wherein protecting is the act of keeping the RNA safe from digestion or cleavage activity from RNases.
  • the poly(A) tail is part of an RNA transcript that is added an RNA transcript (the so called polyadenylation), typically a messenger RNA (mRNA).
  • the poly(A) tail consists of multiple adenosine monophosphates. In eukaryotes, polyadenylation is part of the process that produces mature mRNA for translation.
  • the poly (A) capturing relies on the use of oligonucleotides, preferably DNA oligonucleotides, comprising nucleotide stretches of poly (dT), attached to a solid support (e.g.
  • RNA transcripts typically a messenger RNA (mRNA), wherein the poly(A) tail of the RNA transcript is capable of hybridizing to the poly (dT) nucleotide stretches of the oligonucleotides, preferably by Watson-Crick base paring.
  • mRNA messenger RNA
  • hybridization of two nucleic acid molecules (or oligonucleotides) to each other is through Watson-Crick base pairing.
  • a dried blood spot is generated by spotting at least 50 pl of blood onto a filter paper typically forming part of a filter card.
  • a dried blood spot is generated by transferring at least three blood spots from a blood sample onto a filter paper typically forming part of a filter card.
  • the RNA preparation obtained by the method of the first aspect and the second aspect comprises non-viral RNA such as mammalian and human RNA in particular.
  • said RNA preparation in an embodiment, comprises viral RNA and viral mRNA in particular, if such viral mRNA is contained in the blood or the blood cells (i.e., the first sample).
  • the content of such viral RNA and viral mRNA in particular is reduced so that its content, relative content or absolute content, is decreased compared to its content in the first sample, i.e. the blood and blood cells, respectively, from which the RNA is extracted in accordance with the present invention.
  • an RNA library or an mRNA library are prepared by construction of a cDNA library, followed by DNA library preparation steps: repair of 3' and 5' ends followed by the addition of a non-templated dA-tail before ligation with an adaptor.
  • Libraries are size selected after adaptor ligation, and amplified via polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Small RNA libraries are constructed using a different workflow, in which adaptors are ligated directly to the small RNA molecules, followed by reverse transcription, PCR amplification and size selection. Libraries are then clonally amplified and sequenced using sequencing by synthesis (sbs) methods, such as the Illumina sequencing platform.
  • irregular time intervals are time intervals which do not follow a regular scheme and/or predictable scheme.
  • a predictable scheme is preferably one where a distinct time interval can be predicted based on one or several time intervals preceding said distinct time interval.
  • a typical reason for of the cause of irregular time intervals can be the decision of a physician treating a or the subject to change the treatment in light of any aggravation or improving of the health condition of the subject.
  • a predictive biomarker is a compound, such as nucleic acid molecule, that indicates sensitivity or resistance of a subject to a specific therapy.
  • a prognostic biomarker is a compound, such as a nucleic acid molecule, that can be used to measure the progress of a disease of a subject in a sample from the subject.
  • the RNA Integrity Number is an algorithm for assigning integrity values to RNA measurements.
  • the RNA Integrity Number was developed by Agilent Technologies in 2005 (Schroeder et al., 2005, The RIN: an RNA integrity number for assigning integrity values toRNA measurements, BMC Mol Biol . 2006 Jan 31:7:3. doi: 10.1186/1471-2199- 7-3).
  • the integrity of RNA is a major concern for gene expression studies and traditionally has been evaluated using the 28S to 18S rRNA ratio, a method that has been shown to be inconsistent. This inconsistency arises because subjective, human interpretation is necessary to compare the 28S and 18S gel images.
  • the RIN algorithm was devised to overcome this issue.
  • the RIN algorithm is applied to electrophoretic RNA measurements, typically obtained using capillary gel electrophoresis, and based on a combination of different features that contribute information about the RNA integrity to provide a more universal measure.
  • RIN has been demonstrated to be robust and reproducible in studies comparing it to other RNA integrity calculation algorithms, cementing its position as a preferred method of determining the quality of RNA to be analyzed.
  • the algorithm was generated by taking hundreds of samples and having specialists manually assign them all a value of 1 to 10 based on their integrity, with 10 being the highest.
  • Adaptive learning tools using a Bayesian learning technique were used to generate an algorithm that could predict the RIN.
  • RIN for a sample is computed using several characteristics of an RNA electropherogram trace, with the first two listed below being most significant.
  • RIN assigns an electropherogram a value of 1 to 10, with 10 being the least degraded. All the following descriptions apply to mammalian RNA because RNAs in other species have different rRNA sizes: The total RNA ratio is calculated by taking the ratio of the area under the 18S and 28S rRNA peaks to the total area under the graph, a large number here is desired, indicating much of the rRNA is still at these sizes and thus little to no degradation has occurred.
  • the course of a disease may be determined by the method according to the present invention by determining a expression level of an RNA in the sample from the subject at different time points in the course of the disease. It is important to note that a single application of a method for diagnosing a disease according to the present invention allows for diagnosing the disease and in certain embodiments comprises a step of managing subject treatment based on the diagnosis of whether the subject is suffering from or for being at risk for developing the disease. If a subject a sample of which is thus subjected to the method of the present invention is tested positive for suffering from or to be at risk for developing a disease a skilled clinician will know how to decide concerning managing subject treatment, i.e. how the subject will be treated, e.g. applying a certain dose of a therapeutic or medicament.
  • the skilled clinician may decide for at least one additional application of the method according to the present invention on a later time point. It is thus an embodiment of the present invention that the levels of the RNA (as biomarker) determined at the different time points, wherein different time points means at least two time points, may be compared.
  • the level of the RNA (as biomarker) of the present invention in samples form one particular patient may be correlated to the severity of the disease in said patient at the time point the sample from the patient is taken.
  • an elevated level of the RNA (as biomarker) determined in the sample of a later time point compared to the level of the RNA (as biomarker) determined in the sample of an earlier time point is indicative for a more severe status of the subject at the later time point compared to the status of the subject at the earlier time point.
  • a decreased level of the RNA (as biomarker) determined in the sample of a later time point compared to the level of the RNA (as biomarker) determined in the sample of an earlier time point is indicative for a less severe status of the subject at the later time point compared to the status of the subject at the earlier time point.
  • the present invention provides a method for determining the course of a disease in a subject comprising the step of determining at several points in time a level of an RNA (as biomarker) present in a sample from the subject.
  • the invention concerns a method for determining the effectiveness of at least one treatment applied to a subject being positively tested for suffering from or being at risk for developing a disease comprising the step of determining at several points in time a level of an RNA (as biomarker) present in a sample from the subject.
  • the methods of the present invention thus allow for selecting a therapy and/or adjusting the doses and/or dosage of a selected therapy based on the results of the method of the invention. If for example the subject is scheduled for treating for a disease the method for diagnosing the disease in a subject according to the present invention may be applied every 3 months and levels of the an RNA (as biomarker) thus determined will be compared in order to determine the effectiveness of the treatment(s) and/or therapy/therapies applied to the subject.
  • the frequency of application of the method for diagnosing the disease in a subject according to the present invention may be reduced to every 6 month. If the dosage of the therapy is changed, the frequency of application of the method for diagnosing the disease in a subject according to the present invention may be set back to every 3 month.
  • the skilled physician may decide to reduce the dosage of the therapy, to increase the dosage of the therapy; or to maintain the dosage of the therapy according to the comparison of the levels of the RNA (as biomarker) determined with the method according to the present invention.
  • a reduction of the level of the RNA (as biomarker) or the increase of the level of the RNA (as biomarker) may correlate with the effectiveness of a therapy.
  • the method of the present invention is for comparing the effectiveness of a therapy or of at least two therapies applied to a subject.
  • a person skilled in the art thus will acknowledge that the progression, i.e.
  • the invention concerns a method for determining the effectiveness of at least one treatment applied to a subject being positively tested for suffering from or being at risk for developing a disease comprising the step of determining at several points in time a level of RNA (as biomarker) present in a sample from the subject.
  • a level of RNA as biomarker
  • RNA splicing is a process in molecular biology where a newly- made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). It works by removing all the introns (non-coding regions of RNA) and splicing back together exons (coding regions).
  • pre-mRNA precursor messenger RNA
  • mRNA mature messenger RNA
  • a splice variant (also referred to as splice-site variant or splice-site mutation) according the present invention is a genetic alteration in the DNA sequence that occurs at the boundary of an exon and an intron (splice site). This change can lead to alternative splicing of exons in pre-mRNA resulting in expression of multiple different mature mRNAs (loss of exons or the inclusion of introns and an altered protein-coding sequence) from an individual gene, known as splice variants.
  • Variants may cause changes in the structure of mRNA that affect its splicing, stability, folding and translation efficacy. Many patients remain undiagnosed with variants of unknown significance (VUS) or no relevant variants detected after exome or genome sequencing.
  • VUS variants of unknown significance
  • the effects of VUS on transcript splicing can be variable and depend on the specific location and nature of the variant within the gene. While there are power tools to predict the effects of VUS, studying the RNA expressed with the variants is the confirmation. RNA-seq can be a powerful tool for the diagnosis of genetic diseases. It is particularly useful to assess the impact of variants at the mRNA level. Variant effects on mRNA structure ranges from alternative splicing to decay of the transcript, changes in efficacy of translation to alternation in polyadenylation.
  • RNA splicing is a critical process that generates different mRNA isoforms from a single gene, allowing to produce multiple protein products with diverse functions.
  • RNA-seq data can be used to identify and quantify alternative splicing by analyzing splice junction reads and by examining exon usage. Splice junction reads are generated when RNA-seq reads overlap two adjacent exons, indicating the presence of a spliced transcript isoform. By counting the number of reads that span each splice junction, scientists can quantify the expression levels of different transcript isoforms and identify alternatively spliced exons.
  • Some variants can alter the stability of the mRNA by changing the secondary structure or exposing regions that are more susceptible to degradation by ribonucleases. This can result in differences in the levels of mRNA expression or affect the half-life of the mRNA. Decaying RNA fragments have lower transcript count, thus influencing protein function.
  • a splice junction according to the present invention refers to a pair of 5' and 3' splice sites, which are the boundaries between introns and exons.
  • An allele (also referred to as an allelomorph) according to the present invention, is a variant of the sequence of nucleotides at a particular location, or locus, on a DNA molecule. Alleles can differ at a single position through single nucleotide polymorphisms (SNP), but they can also have insertions and deletions of up to several thousand base pairs.
  • SNP single nucleotide polymorphisms
  • a data bank (also referred to as databank) according to the present invention is a repository of information about one or more subjects, that is, a database which is organized in a way that facilitates local or remote information retrieval and is able to process many continual queries over a long period of time.
  • Transcription according to the present invention is the process of copying a segment of DNA into RNA.
  • the segments of DNA transcribed into RNA molecules that can encode proteins produce messenger RNA (mRNA).
  • Other segments of DNA are transcribed into RNA molecules called non-coding RNAs (ncRNAs).
  • ncRNAs non-coding RNAs
  • a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand called a transcript of a gene.
  • the method according to the present invention can be used to identify a portion or population of subjects with a specific RNA expression profile.
  • Relative abundance is a measure of how common or rare a species is relative to other species in a defined population. Relative abundance is the percent composition of a species of a particular kind relative to the total number of species in the analyzed population.
  • a metabolic pathway according to the present invention is a linked series of chemical reactions occurring within a cell.
  • the reactants, products, and intermediates of an enzymatic reaction are known as metabolites, which are modified by a sequence of chemical reactions catalyzed by enzymes.
  • the product of one enzyme acts as the substrate for the next.
  • side products are considered waste and removed from the cell.
  • RNA-seq can measure the sum of variant mechanisms controlling gene expression levels: Epigenetic modifications, RNA process machinery, RNA stability, Regulatory elements and cellular signaling. The change in one gene expression is usually accompanied by changes in related pathways.
  • RNA-seq can be used to measure pathway enrichment by comparing the expression of genes in a particular pathway to the expression of genes in the background of all expressed genes in DBS.
  • Gene expression quantification is performed, then statistical gene set enrichment analysis using existing pathway databases.
  • RNA-seq to measure pathway enrichment, insights can be gained into the biological processes that are altered in a particular sample, such as the effects of a drug treatment, disease state, or environmental exposure. This can help to identify potential therapeutic targets and biomarkers, and aid in the development of personalized medicine.
  • Pathway enrichment analysis requires a high number of samples to uncover underlying causes of rare diseases. The most practical way to collect high numbers of RNA samples is using DBS.
  • GSEA gene set enrichment analysis
  • the human genome is diploid, with each individual generally carrying two copies of each chromosome.
  • Each chromosome harbors one copy of each gene, referred to as allele, each of which is inherited by one of the two parents.
  • the two gene alleles are generally expressed at similar levels in a tissue, but cis-regulatory differences between them, for example, differential binding of transcription factors (TFs), can lead to systematic differences between the expression of the two alleles in an individual. This is commonly referred to as allelic imbalance or allele-specific expression.
  • allelic imbalance or allele-specific expression In extreme instances, only one copy of the gene is expressed, a phenomenon called mono-allelic expression, that can be due to gene deletion or to epigenetic mechanisms such as imprinting or X chromosome inactivation.
  • the gene expression measured for each gene allele individually also called allelic expression quantification, can be generated using RNA sequencing data and allows to quantify the degree of allelic imbalance in each gene.
  • the method comprises the preparation of an RNA library from the RNA preparation.
  • RNA library preparation comprises fragmentation and priming of the RNA from the RNA preparation, wherein after fragmenting the RNA into fragmented libraries cDNAs thereof are prepared that are ligated to adapters to allow library amplification and strand selection.
  • a detailed protocol can be found in the Example section.
  • sequencing of RNA amplifying of RNA, determining and quantifying the amount of RNA is carried by High-throughput sequencing (abbr. HTS) methods which is also refered to as "Next-Generation” or “Second-Generation” sequencing (abbr. NGS) or Quantitative PCR (formally quantitative real-time PCR, qPCR).
  • HTS High-throughput sequencing
  • NGS Next-Generation sequencing
  • Quantitative PCR quantitative real-time PCR
  • Quantitative PCR (formally quantitative real-time PCR, qPCR) detection builds on the basic PCR technique and allows researchers to estimate the quantity of starting material in a sample. Since the products are detected as the reaction proceeds, qPCR has a much wider dynamic range of analysis than conventional, end-point PCR; from a single copy to around 10 11 copies are detectable within a single run. Quantitative real-time PCR and subsequent amplicon detection is carried out in a closed-tube format which eliminates the need for post-PCR manipulation, such as gel electrophoresis and significantly reduces the risk of cross contamination.
  • a fluorescent reporter dye is used as an indirect measure of the amount of nucleic acid present during each amplification cycle.
  • reporter molecules are categorized as; double-stranded DNA (dsDNA) binding dyes, dyes conjugated to primers, or additional dye-conjugated oligonucleotides, referred to as probes (Quantitative PCR and Digital PCR Detection Methods).
  • dsDNA double-stranded DNA
  • probes additional dye-conjugated oligonucleotides
  • SYBR® Green I represents the simplest form of detection chemistry. When free in solution or with only single-stranded DNA (ssDNA) present, SYBR Green I dye emits light at low signal intensity.
  • a probe (or combination of two depending on the detection chemistry) can add a level of detection specificity beyond the dsDNA-binding dye, since it binds to a specific region of the template that is located between the primers.
  • the most commonly used probe format is the Dual-Labeled Probe (DLP; also referred to as a Hydrolysis or TaqMan® Probe).
  • the DLP is an oligonucleotide with a 5’ fluorescent label, e.g., 6-FAMTM and a 3’ quenching molecule, such as one of the dark quenchers e.g., BHQ®1 or OQTM . These probes are designed to hybridize to the template between the two primers and are used in conjunction with a DNA polymerase that has inherent 5’ to 3’ exonuclease activity. When the DLP is free in solution, the signal intensity is low because the reporter dye is in close proximity to the quencher moiety.
  • a 5’ fluorescent label e.g., 6-FAMTM
  • a 3’ quenching molecule such as one of the dark quenchers e.g., BHQ®1 or OQTM .
  • probes labeled with different reporter dyes allows for the simultaneous detection and quantification of multiple targets in a single (multiplex) reaction.
  • a typical qPCR run consists of repeated cycles of alternating temperature incubations. This profile is often used when dsDNA- binding dyes, Molecular Beacons, or Scorpion® Probes are the chosen detection chemistries for qPCR. Primer extension is most efficient at 72 °C because this is the optimal temperature for processivity of most DNA polymerases.
  • polymerization occurs at a rate of approximately 100 bases per second.
  • processivity at lower temperatures that is sufficient to amplify shorter templates.
  • qPCR amplicons are typically shorter ( ⁇ 200 bases) than conventional PCR products, thus extension is often combined with annealing in a single step at 60 °C when working with Dual-Labeled Probles.
  • High-throughput sequencing (HTS) methods also referred to as “next-generation” or “second- generation” sequencing (NGS) are typically characterized by being highly scalable, allowing the entire genome to be sequenced at once. Usually, this is accomplished by fragmenting the genome into small pieces, randomly sampling for a fragment, and sequencing it using one of a variety of technologies. An entire genome is possible because multiple fragments are sequenced at once (giving it the name “massively parallel” sequencing) in an automated process.
  • RNA sequencing This method, termed RNA sequencing (abbr. RNA-Seq), has eliminated many challenges posed by hybridization-based microarrays and Sanger sequencingbased approaches that were previously used for measuring gene expression and has revolutionized the understanding of the complex and dynamic nature of the transcriptome.
  • RNA-Seq provides a more detailed and quantitative view of gene expression, alternative splicing, and allele-specific expression.
  • Advances in the RNA-Seq workflow from sample preparation to sequencing platforms to bioinformatic data analysis, has enabled deep profiling of the transcriptome and the opportunity to elucidate different physiological and pathological conditions.
  • After sequencing the data are routinely processed to generate the gene counts which are the basis for all subsequent bioinformatic RNA sequencing analyses. Generating gene counts is based on known (publicly available) pipelines for NGS analysis.
  • Fig. 1 shows dried blood spots on a blood filter card prepared from lOOpl of EDTA blood
  • Fig. 4 shows a boxplot showing the RNA yield [ng] of RNAs obtained from a total of 583 filter cards with a card age between 1 day and 14 days, wherein the filter cards were stored at -80°C prior to RNA extraction; RNA concentrations were assessed with the Qubit HS RNA assay;
  • Fig. 6 shows fraction of samples of RNA obtained from filter cards with a RIN > 2.5 (dark grey) and RIN ⁇ 2.5 (light grey) over the card age [d]; the line is showing the percentage of cards with a RIN > 2.5; RIN values assessed with Agilent Tapestation RNA HS Screentape.
  • Fig. 8 shows gene body coverage for RNA samples obtained from dried blood spots; the graph shows the 5' - 3' coverage of the samples, where a shift to the 3' end indicates higher RNA degradation; the data reflects the results from a protocol version only using Poly(A)-selection and globin depletion;
  • Fig. 9 shows gene body coverage for RNA samples obtained from dried blood spots; the graph shows the 5' - 3' coverage of the samples, where a shift to the 3' end indicates higher RNA degradation; the data presented here were obtained with the protocol presented herein (see Example 1, Example 4 and Example 6);
  • Fig. 13 shows a Pie chart showing the results of 103 patients with a predicted/potential splice variant identified in whole exome/ whole genome sequencing that were reanalyzed by RNA seq of RNA obtained from DBS to confirm/reject an effect on the RNA transcript.
  • *Likely abnormal splicing ⁇ 5 supporting reads;
  • Fig. 14 shows a Sashimi plot (IGV) showing a heterozygous variant in the DYSF gene (c.5785- 824OT); the deep intronic variant results in intron retention in form of a cryptic exon;
  • Fig. 15 shows a sashimi plot (IGV) showing a homozygous variant in the DNAJB2 gene (c.176- 2A>G).
  • the canonical splice site variant leads to the skipping of Exon04 of the transcript;
  • Fig. 16 shows Sashimi plot (IGV) showing a heterozygous variant in the SBDS gene (c.258+2T>C) leading to an altered splice site in some of the reads;
  • Fig. 17 shows IGV screenshot of the patient presented in Figure 15; some transcripts show a deletion of the last 8 bases in Exon 02 of the SBDS gene;
  • Fig. 18 shows a gene set enrichment analysis in a Parkinson disease cohort vs controls whereby a positive or negative enrichment score (ES) indicates gene set enrichment;
  • Example A) shows the MTOR signaling pathway enriched in the patient cohort carrying a GBA variant;
  • Example B) shows negative regulation of the lysosome pathway in the same cohort;
  • Fig. 19 shows a IGV screenshot showing the allele penetrance of heterozygous GBA mutation N409S wherein the example is showing a balanced allele frequency
  • Fig. 20 shows a IGV screenshot showing the allele penetrance of a heterozygous GBA mutation N409S; the example is showing an imbalanced allele frequency (70:30);
  • Fig. 21 shows clustering results of time course analysis of RNA seq data showing upregulation of a gene cluster at timepoint Post 1 (Prel and Pre2 data were merged to one timepoint);
  • Fig. 22 shows pathway enrichment for Cluster 1 (presented in Figure 21) using ToppGene showing upregulation in the interferon alpha/beta signaling pathway;
  • Fig. 23 shows normalized counts (DESeq2) from DBS samples of individuals before and after covidl9 vaccination (prel: 3-4 days prior vaccination, pre2: 1-2 days prior, postl: 1-2 days after vaccination, post2: 3-4 days after, post3: 5-7days after); time course analysis showed the upregulation of genes from the interferon alpha/beta pathway as an early response to the vaccine (here: EIF2AK2 and IFI35); and
  • Fig. 24 shows normalized counts (DESeq2) from DBS samples of individuals before and after covidl9 vaccination (prel: 3-4 days prior vaccination, pre2: 1-2 days prior, postl: 1-2 days after vaccination, post2: 3-4 days after, post3: 5-7days after); time course analysis showed the upregulation of genes from the interferon alpha/beta pathway as an early response to the vaccine (here: IRS2 and MAP4K4).
  • a dry blood spot (abbr. DBS) on a filter card was used as a sample from a subject.
  • the filter paper of the filter card is the Ahlstrom 226 filter paper. Specifications of the Ahlstrom 226 filter paper can be found within the document. Nevertheless, a person skilled in the art will acknowledge that depending on the used type of sample from a subject, e.g. comprising saliva, cerebrospinal fluid, plasma, serum, full blood, blood on a dry blood filter card or another blood product, the method of the present invention has to be adjusted to the type of sample according to the method described in the following examples.
  • a sample was collected using the VacutainerTM Plastic K2EDTA (BD). The tube was inverted 10 times to ensure mixing of EDTA and blood. 50 to lOOpl of blood were dropped onto each spot of the filter card (Fig. 1). The cards were dried open for 2 hours at room temperature. Afterwards the filter cards were kept in a plastic envelope at room temperature until further processing. How the blood was collected and which amount of blood was spotted on the filter is specified in each example.
  • RNA Column Zymo Spin Column
  • Zymo R1051 RNA Column
  • the flow throw was discarded and 400 pl RNA Wash Buffer (Zymo R1051) was added to the column, then tube with column centrifuged for 30 sec at 16.000 ref and the flow through discarded. 40pl DNasel Digestion Mix was added to the column and incubated for 15 min at room temperature.
  • RNA High Sensitivity buffer 1 pl RNA High Sensitivity buffer was mixed with 2 pl sample. The mixture was incubated 3 minutes at 72°C and run with an RNA High Sensitivity Screen tape on the Agilent Tapestation to determine the RNA integrity number (RIN).
  • the Proteinase K Master Mix contains a DNAse and RNAse inhibitor, e.g. DNA/RNA shield (Zymo Research) and proteinase K to lyse the cells and inactivate RNAses to prevent RNA degradation.
  • RNAse inhibitor e.g. DNA/RNA shield (Zymo Research) and proteinase K to lyse the cells and inactivate RNAses to prevent RNA degradation.
  • RNA from dried blood spots EDTA blood
  • the stability was tested over a period of 21 days.
  • 2 spots from 2 different filter cards, prepared according to Example 1 were cut out with a scalpel and RNA was extracted.
  • the RNA extractions for this experiment were deviating from the described protocol (see Example 1) in the sense that each spot was incubated individually in a 2ml tube at 37°C and 1500rpm with 400pl of the Proteinase-K-Mix.
  • Figure 2 shows the results of the stability test.
  • the RIN remained stable over the first 3 days. After 4 days the RIN was decreased to 6 and it stayed stable until day 10. After 14 days we observed a RIN of 4 and after 21 days the RIN value was further reduced to 3.
  • RNA of high quality can be extracted from dried blood spots over a period that makes it available for diagnostic settings, where shipment of filter cards often takes several days to 2 weeks.
  • 2 filter cards were prepared according to the description in Example 1 and kept at room temperature for 8 days. After 8 days 12 spots from the filter cards were cut out with a scalpel. For each timepoint (1, 2 and 3 month(s)) 4 spots were stored in a 2ml tube at -80°C. After the respective times RNA was extracted from these spots.
  • the RNA extractions for this experiment were deviating from the described protocol (see Example 1) in the sense that 4 spots were used where each spot was incubated individually in a 2ml tube at 37°C and 1500rpm with 400 pl of the Proteinase-K-Mix. After the incubation and addition of lx volume 100% ethanol to each tube the whole volume of the tubes was added stepwise to one Zymo spin column and centrifuged according to the standard protocol described in Example 1. Furthermore, the elution volume of 25 pl of nuclease-free water differed from the standard protocol described in Example 1.
  • Figure 3 shows the results of the stability at -80°C experiment. During the 3 months of storage at -80°C the RIN value of the sample showed only a slight decrease from 5.3 to 4.4. The storage of the filter cards at -80°C prevented further degradation of the RNA on the filter card and the integrity could also be preserved during the extraction procedure.
  • the presented data show that with the herein described protocol RNA of high quality can be extracted from dried blood spots over a period that makes it available for diagnostic settings, for example for samples that were stored for several months at -80°C.
  • Example 3 Characteristics of extracted RNAs from real-life samples
  • RNA from dried blood spots of consented patients was extracted to investigate the usability of RNA from DBS in a real-life diagnostic setting.
  • RNA yield of 583 DBS samples was stable over the examination period of 14 days.
  • RNA yield for more than 99% of the cards was more than 25 ng after extraction which according to the extraction protocol described herein (see Example 1), was tested as the minimum required amount for RNA library preparation (see Example 5).
  • the presented data show that with the herein described protocol RNA of high quality can be extracted from dried blood spots over a period that makes it available for diagnostic settings, where shipment of filter cards often takes several days to 2 weeks.
  • RNA quality can be achieved from finger prick samples. Therefore, up to 6 blood drops were put onto one filter card spot with 2 spots in total. The filter cards were dried for 24 hours at room temperature. Afterwards extraction was performed according to Example 1. Table 1 shows the results of the extraction. Due the good RNA quality of the finger prick samples an RNA library was generated as described in Example 4. Afterwards the sample was sequenced (see Example 6). Sequencing results were compatible with results from the EDTA blood filter cards. This allows a more convenient way of sample collection, especially for repeated sampling as in longitudinal studies.
  • Table 2 RIN and RNA amount extracted from 2 dried blood spots prepared from finger prick of 3 individuals. Sample RIN RNA yield [ng]
  • 25ng - 50ng total RNA was diluted in nuclease-free water to a volume of lOpl and incubated in a thermocycler at 60°C for 1 min. Until the beads were added the sample was kept at 25°C.
  • Opl of denatured RNA was added to 10 pl of washed beads and incubated in a thermomixer at 25°C for 20 min with 1250rpm. The plate was transferred onto a magnetic rack and incubated until the supernatant was clear and was removed. The plate was removed from the rack and lOOpl BWB was added. The beads were resuspended and incubated on the thermomixer at 25°C for 5 min with 1250rpm. Afterwards the beads were collected on the magnet until the supernatant was clear and removed. The washing step were repeated for a total 2 washes and the end the supernatant was completely removed.
  • RNA was eluted from the Poly(A) beads with 20 pl nuclease free water and incubation at 70°C for 1 min. The plate was immediately placed on the magnet until the supernatant was clear. 18 pl of the supernatant was transferred to the corresponding well of a new 96 well PCR plate for further processing.
  • rRNA and hemoglobin RNA depletion rRNA and Globin Depletion
  • the Depletion Reaction Mix was prepared (on ice).
  • the sealed PCR plate was centrifuged and 35 pl of DNase Master Mix was added to the depleted RNA.
  • the sealed plate was vortexed, centrifuged and placed on a thermal cycler to run the following DNase Digestion Program.
  • the Frag & Prime Master Mix was prepared as follows for each reaction.
  • the plate was placed on the thermal cycle and the program Fragmentation program was run. After the program was finished, the plate was placed on a magnet until all beads were collected on the tube wall and the solution was clear. 25 pl of the clear supernatant containing the fragmented libraries are transferred into the corresponding well of a new 96 well plate that contains 10 pl of pre-aliquoted 1st Strand Synthesis Master Mix. After mixing the was placed in thermocycler and the 1st Strand Synthesis Program was started.
  • 2nd Strand Master Mix was prepared as follows:
  • the thawed Ligation Buffer was vortexed for 20 sec to fully homogenize the solution.
  • the truncated “stubby” adapters (IDT) are diluted from 15pM to 0.4 pM. 5 pl of the diluted adapters were added to each well containing the 2nd strand mix.
  • Ligation Master Mix was prepared as follows:
  • the Ligation Master Mix was then vortexed for another 20 sec to thoroughly mix the reagents. 45 pl of the Ligation Master Mix were added to each well and mixed by pipetting for 10 x with the pipette set to 80pl. The plate was then placed in a thermocycler and the Adapter Ligation Program was started.
  • the plate was removed from the magnet and each bead pellet was resuspended in 22 pL nuclease-free water. The plate was incubated for 2 min at room temperature, before placing it back on the magnet until the solution was clear. Then 20 pL of the purified sample were transferred to a new plate.
  • the plate was placed in a thermocycler and the Library Amplification program was initiated.
  • 80% EtOH was freshly prepared (0.4ml for each reaction.
  • the Ampure XP Beads were equilibrated to room temperature and mixed by vortexing. 50 pl Ampure XP Beads were added to each well. The plate was sealed and mixed on a vortexer followed by 5 min incubation at room temperature. After that the plate was placed on a magnet until the solution was clear. The supernatant was removed from each well and 200 pl freshly prepared 80% ethanol was added to each well, incubated 30 sec and removed. The washing step was repeated for a total of two washes. Then the EtOH was removed and evaporated for 3 min at RT.
  • the plate was removed from the magnet and each bead pellet was resuspended in 22 pL nuclease-free water. The plate was incubated for 2 min at room temperature, before placing it back on the magnet until the solution was clear. Then 20 pL of the purified sample were transferred to a new plate.
  • the library was checked with a DNA 1000 Screentape on the Tapestation (Agilent).
  • the Tapestation profiles were inspected for the average library size to be around 320 - 420 bp without adapter dimer peaks (-200 bp).
  • the libraries were quantified using the 1 x DNA High Sensitivity assay and the Qubit Flex (Life Technologies). The nanomolarity of each library was calculated based on the average size (Tapestation) and the concentration (Qubit) according to the following formula:
  • the libraries were pooled equimolarly, and the concentration of the final pool was measured using the Qubit 1 x DNA High Sensitivity assay and the Qubit (Life Technologies). The final pool was then sequenced using Illumina sequencing technology.
  • RNA yield especially for filter cards from the real-life setting, can sometimes be low, we tested different total RNA input amounts for the library preparation. Test criteria were the successful generation of a library and that our general sequencing quality, i.e. total number of paired reads >20M, unique assigned reads >4M unique assigned reads, number of genes >8000. We tested low (25ng), medium (50ng) and high (lOOng) RNA inputs from dried blood spots.
  • Example 1 The samples were prepared according to the protocols (Example 1, Example 4, Example 6) and the correlation of normalized read counts for the different input amounts and samples was calculated.
  • the final pool was sequenced on a NextSeq 500 or NovaSeq 6000 device (both Illumina).
  • the NextSeq 500 the High Outut Kit v2.5 was used. Up to 30 samples were pooled for a sequencing run on the NextSeq.
  • the sequencing on the NovaSeq 6000 the SI Reagent Kit vl.5. On the SI flow cell up to 96 samples in total were pooled for one run.
  • the 75 paired-end protocol refers to a sequencing length of 75 bases, where every fragment is sequenced from both sides resulting in two reads per fragment.
  • the alternative protocol tested was the 150 paired-end protocol that generates longer reads. The average total number of reads over all runs was 20 M paired reads.
  • Generating gene counts is based on known (publicly available) pipelines for Illumina NGS analysis. Briefly, the photos generated from the sequencer (bcl files) were converted to text sequence (fastq file) using bcl2fastq tool. STAR2.7 was used to align the reads to the human reference genome (hgl9), thenPCR duplicates were removed (e.g. SAMtools and PICARD tools). Read counts were generated via counting tools (e.g. HTSseq and featureCount), normalized counts were generated by normalization tools (e.g. Stringtie2, STAR2.7, DESeq2). Bioinformatics QC was generated using several RNA-seq QC tools (FastQC, MultiQC, RNA-QC). Samples below QC cut-off (total number of reads >20M, number of unique assigned reads > 4M) were removed from further analysis. Samples were then further normalized, if necessary, based on the experimental design (by Gender, Age etc.)
  • differential gene expression is performed by tools like DESeq2 or EdgeR.
  • a list of genes is obtained that have statistically different gene expression in patients than healthy, or in treated than untreated or any other two conditions. This list of genes is investigated for enriched pathways in tools such as Toppg ene, PantherDB or David.
  • GSEA gene set enrichment analysis
  • RNA-seq data can be analyzed for changes with time (different time points- longitudinal study) or with different doses (response to therapy). They can be called time course analysis among other labels.
  • Many tools can be used to do time course analysis including TCseq, DESeq2 and DEGreport.
  • Figure 7 shows the results obtained from the first protocol version that was only based on the extraction protocol according to Example 1, a Poly(A) selection (Illumina stranded mRNA Kit) and a subsequent globin gene depletion (QIAseq Fast Select globin Kit).
  • the result presented in Figure 7 is showing a high correlation between blood RNA and DBS RNA of 0,91.
  • RNA yield was below 50 ng for a high number of samples.
  • the final library preparation protocol (see Example 4) only required a minimum of 25 ng RNA.
  • a Poly(A) selection and subsequent rRNA/globin depletion was still showing high correlations between Blood and DBS samples even though less total RNA starting material was used (see Figure 10).
  • the result presented in Figure 10 is showing a high correlation between blood RNA and DBS RNA of 0,95 (slightly improved in comparison to the first protocol version).
  • RNA analysis from blood is the PAXgene Blood tube that contains a reagent that is preventing RNA degradation. Therefore, it was also tested how high the gene expression correlation was between DBS RNA obtained with the herein described protocol (Example 1 and Example 4) and RNA obtained from PAXgene Blood RNA tubes using the PAXgene Blood RNA Kit.
  • the result presented in Figure 11 is showing a high correlation between PAXgene blood RNA and DBS RNA of 0.87 and 0.88, respectively.
  • the data quality of RNA sequencing from dried blood spots is comparable to RNA extracted from commonly used blood collection products, e.g, PAXgene RNA blood or EDTA blood.
  • RNA-seq from blood covers around 43% of the OMIM genes (genes with a known phenotype-causing mutation) with good coverage (Reads Per Kilobase Million- RPKM > 4).
  • Figure 12 shows that mRNA sequencing from dried blood spots covered almost the same number of OMIM genes (-41%). The result presented in Figure 12 is showing a high correlation for gene coverage between blood RNA and DBS RNA.
  • RNA filter cards resulted in a broad range of RIN values.
  • RIN Random Access to N-(A) selection
  • Samples with such low RIN values are suspected to be highly degraded and would potentially decrease the coverage of 5’ end of transcripts (PMID: 24632678).
  • the gene body coverage was less affected than expected from literature as shown in Figure 8 (results obtained from the first protocol version that was only based on the extraction protocol according to Example 1, a Poly(A) selection (Illumina stranded mRNA Kit and a subsequent globin gene depletion (QIAseq Fast Select globin Kit)) and Figure 9 (improved protocol according to Example 1 and Example 4).
  • RNA obtained from blood samples e.g. EDTA blood samples
  • DBS using the protocol according to example 1 and 4
  • RNA preparation from DBS using the protocol according to example 1 and 4 can be used in diagnostics.
  • Variants may cause changes in the structure of mRNA that affect its splicing, stability, folding and translation efficacy. Many patients remain undiagnosed with variants of unknown significance (VUS) or no relevant variants detected after exome or genome sequencing.
  • VUS variants of unknown significance
  • the effects of VUS on transcript splicing can be variable and depend on the specific location and nature of the variant within the gene. While there are power tools to predict the effects of VUS, studying the RNA expressed with the variants is the confirmation. RNA-seq can be a powerful tool for the diagnosis of genetic diseases. It is particularly useful to assess the impact of variants at the mRNA level.
  • Variant effects on mRNA structure ranges from alternative splicing to decay of the transcript, changes in efficacy of translation to alternation in polyadenylation.
  • RNA splicing is a critical process that generates different mRNA isoforms from a single gene, allowing to produce multiple protein products with diverse functions.
  • RNA-seq data can be used to identify and quantify alternative splicing by analyzing splice junction reads and by examining exon usage. Splice junction reads are generated when RNA-seq reads overlap two adjacent exons, indicating the presence of a spliced transcript isoform. By counting the number of reads that span each splice junction, scientists can quantify the expression levels of different transcript isoforms and identify alternatively spliced exons.
  • Some variants can alter the stability of the mRNA by changing the secondary structure or exposing regions that are more susceptible to degradation by ribonucleases. This can result in differences in the levels of mRNA expression or affect the half-life of the mRNA. Decaying RNA fragments have lower transcript count, thus influencing protein function.
  • RNA sequencing 103 patients for whom splicing variants had been reported were selected for RNA sequencing, considering the gene expression in blood and sample availability.
  • the filter cards were kept at - 80°C for different periods of time (up to 3 month) and RNA was extracted (see Example 1).
  • RNA libraries were generated (see Example 4) and sequencing on Illumina NextSeq500 or NovaSeq 6000 device was performed, followed by a data processing including alignment to the reference genome hgl9 (see Example 6).
  • the confirmed abnormal splicing effects comprised: i) intron inclusion, ii) exon(s) skipping and iii) partial exon deletion.
  • Figure 14 results are presented for the observed splicing effects.
  • Figure 13 shows the Sashimi plot of a heterozygous, deep intronic variant in the DYSF gene (c.5785-824OT) resulting in an additional splice site causing the inclusion of the intron (formation of a cryptic exon).
  • a homozygous splice site variant in the DNAJB2 gene is presented (c,176-2A>G).
  • the Sashimi plot shows that Exon 04 is skipped in all reads leading to a shorter transcript.
  • Figure 15 and Figure 16 a heterozygous variant in the gene SBDS (c.258+2T>C) is shown.
  • the splice site variant causes partial loss of the canonical splice donor site.
  • RNA-seq can measure the sum of variant mechanisms controlling gene expression levels: Epigenetic modifications, RNA process machinery, RNA stability, Regulatory elements and cellular signaling.
  • RNA- seq can be used to measure pathway enrichment by comparing the expression of genes in a particular pathway to the expression of genes in the background of all expressed genes in DBS.
  • Gene expression quantification is performed, then statistical gene set enrichment analysis using existing pathway databases.
  • RNA-seq By using RNA-seq to measure pathway enrichment, insights can be gained into the biological processes that are altered in a particular sample, such as the effects of a drug treatment, disease state, or environmental exposure. This can help to identify potential therapeutic targets and biomarkers, and aid in the development of personalized medicine.
  • Pathway enrichment analysis requires a high number of samples to uncover underlying causes of rare diseases.
  • the most practical way to collect high numbers of RNA samples is using DBS.
  • GSEA gene set enrichment analysis
  • RNA extraction (see Example 1), library preparation (see Example 4) and sequencing (see Example 6) were performed for a total of 365 samples.
  • Gene set enrichment analysis was performed for the group of Parkinson patients carrying a GBA variant and control samples.
  • Figure 18 is showing 2 examples of the GSEA results.
  • the upregulation of MTOR signaling pathway and downregulation of the lysosome pathway has already been described in the literature (PMID: 27263112, PMID: 30744070).
  • Allele penetrance refers to the degree to which a genetic variant (allele) is expressed in an individual's phenotype. There are some cases in which it may be possible to detect allele penetrance from RNA-seq data, particularly if the genetic variant in question is located within a coding region of a gene and produces a transcript that can be distinguished from other transcripts. In these cases, it may be possible to use RNA-seq data to compare the expression of the two alleles and to determine the degree to which each allele contributes to the overall phenotype.
  • Samples for examining the allelic balance were prepared according to Example 1, a Poly(A) selection (Illumina stranded mRNA Kit), subsequent globin gene depletion (QIAseq Fast Select globin Kit) and Example 6 ( Figure 19) or to Example 1, Example 4 and Example 6 ( Figure 20).
  • the bam files aligned to hgl9 were visually inspected via IGV.
  • Figure 19 and Figure 20 present a heterozygous variant in the GBA gene.
  • the allele frequency observed in IGV shows a balanced frequency for the patient shown in Figure 19.
  • Figure 20 is an example for allelic imbalance.
  • the heterozygous variant has a frequency of 70% on the mRNA level.
  • the presented data prove that the herein described protocols produce a data quality that is sufficient to determine the degree to which a genetic variant (allele) is expressed in an individual's phenotype.
  • RNA-seq can also be used to gain insights into the underlying molecular mechanisms of rare diseases, which can inform the development of new therapies and treatments. By identifying key genes and pathways that are dysregulated in a rare disease, researchers can develop targeted therapies that aim to correct these defects and improve patient outcomes.
  • RNA-seq can be used to measure gene expression changes over time, which can be particularly useful for monitoring the effects of treatments on gene expression. For example, in a study of treated patients with a particular disease, DBS samples can be collected from patients before treatment, and then at regular intervals after treatment. By analyzing the RNA-seq data from these samples, changes can identify in gene expression levels that are associated with the treatment.
  • RNA from dried blood spots we selected 6 consented individuals that were about to get their 2 nd covid 19 vaccination (Pfizer/B iontech or Moderna). Blood collection, filter card preparation and RNA extraction were conducted as described in Example 1. The generation of mRNA libraries was performed according to Example 4. Sequencing and data processing according to Example 6.
  • Clustering genes in time course analysis can be performed via a variety of tools including TCSeq. Normalized gene expression can be obtained from DESeq2 normalized counts function.
  • the presented data prove that the herein described protocols produce a data quality that is sufficient to measure gene expression changes over time, which can be particularly useful for monitoring the effects of treatments on gene expression.

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Abstract

La présente invention concerne un procédé de préparation d'ARN comprenant un ARN cible, le procédé incluant : - une étape d'extraction d'un ARN à partir d'un premier échantillon, le premier échantillon étant un échantillon sanguin séché, et l'étape d'extraction présente un deuxième échantillon comprenant l'ARN extrait ; - une étape d'épuisement d'un ARN non cible à partir du deuxième échantillon, l'ARN non cible est choisi dans le groupe comprenant l'ARN de l'hémoglobine, l'ARNr et une combinaison d'ARN de l'hémoglobine et d'ARNr, l'étape d'épuisement présente un troisième échantillon comprenant l'ARN cible et duquel l'ARN non cible est appauvri ; et éventuellement - une étape d'enrichissement de l'ARN cible dans le troisième échantillon ou à partir de celui-ci, l'étape d'enrichissement fournissant un quatrième échantillon comprenant l'ARN cible enrichi, l'étape d'extraction d'un ARN à partir d'un premier échantillon comprenant des mesures de protection de l'ARN contre la dégradation et de digestion des protéines contenues dans le premier échantillon, si le premier échantillon comprend un compartiment particulaire de sang comprenant des cellules, les cellules du premier échantillon sont lysées pour libérer l'ARN des cellules avant ou concomitamment à l'extraction d'un ARN du premier échantillon, et le troisième échantillon et/ou les quatrièmes échantillons constituant(nt) la préparation d'ARN comprenant un ARN cible.
PCT/EP2024/054459 2023-02-21 2024-02-21 Procédé de préparation d'une préparation d'arn et son utilisation Ceased WO2024175682A1 (fr)

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EP23157875 2023-02-21
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US63/491,546 2023-03-22

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5654179A (en) * 1990-11-14 1997-08-05 Hri Research, Inc. Nucleic acid preparation methods

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5654179A (en) * 1990-11-14 1997-08-05 Hri Research, Inc. Nucleic acid preparation methods

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BYBJERG-GRAUHOLM JONAS ET AL: "RNA sequencing of archived neonatal dried blood spots", MOLECULAR GENETICS AND METABOLISM REPORTS, vol. 10, 1 March 2017 (2017-03-01), pages 33 - 37, XP055819459, ISSN: 2214-4269, DOI: 10.1016/j.ymgmr.2016.12.004 *
JINSUNG JANG ET AL: "Comparative evaluation for the globin gene depletion methods for mRNA sequencing using the whole blood-derived total RNAs", BMC GENOMICS, BIOMED CENTRAL LTD, LONDON, UK, vol. 21, no. 1, 11 December 2020 (2020-12-11), pages 1 - 9, XP021285517, DOI: 10.1186/S12864-020-07304-4 *
REUST MARY J. ET AL: "Dried Blood Spot RNA Transcriptomes Correlate with Transcriptomes Derived from Whole Blood RNA", THE AMERICAN SOCIETY OF TROPICAL MEDICINE AND HYGIENE, vol. 98, no. 5, 9 May 2018 (2018-05-09), US, pages 1541 - 1546, XP093116970, ISSN: 0002-9637, DOI: 10.4269/ajtmh.17-0653 *
SCHROEDER ET AL.: "The RIN.- an RNA integrity number for assigning integrity values to RNA measurements", BMC MOL BIOL, vol. 7, 2005, pages 3
ZHAO SHANRONG ET AL: "Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion", SCIENTIFIC REPORTS, vol. 8, no. 1, 19 March 2018 (2018-03-19), pages 4781, XP055982243, Retrieved from the Internet <URL:https://www.nature.com/articles/s41598-018-23226-4.pdf> DOI: 10.1038/s41598-018-23226-4 *

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