Editing profiling of PDE8A pre-mRNA: use as specific biomarker of ADARs activities in human tissues to diagnose and to predict and assess therapeutic efficacy and/or efficiency or potential drug side effects

10851,420

16/442825

June 17, 2019

The present invention relates to the use of the editing profile of PDE8A pre-mRNA as a specific bio marker of ADARs activities in evolved primate, particularly in Human tissues. The present invention also relates to an in vitro method for predicting in Human an alteration of the mechanism of the ADARs catalysed pre-mRNA editing of target genes, by analysing the PDE8A pre-mRNA editing profile in a peripheral tissue sample containing cells expressing said PDE8A pre-mRNA, such as blood sample. The present invention is also directed to an in vitro method for the screening of potential therapeutic compound and to predict and assess therapeutic efficacy and/or efficiency or to diagnose potential severe brain or peripheral drug side effects implementing said PDE8A pre-mRNA editing profile as specific biomarker. The present invention is further directed to a method for determining the PDE8A pre-mRNA editing profile in Human, particularly by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) method after amplification by a nested PCR. Finally the invention relates to particular nucleic acid primers implemented in said nested PCR and kit comprising such sets of primers and human cells capable of expressing PDE8A and ADARs.

ALCEDIAG (Peynier, FR)

ALCEDIAG (Peynier, FR)

1. An in vitro method, comprising: a) providing a biological subject sample comprising Peripheral Blood Mononuclear Cells (PBMC), said PBMC expressing the editing enzymes ADAR1a, ADAR1b and ADAR2, and the phosphodiesterase subtype 8A (PDE8A); b) preparing a cellular RNA extract from the subject sample; and c) determining the editing profile of the PDE8A pre-mRNA in said cellular RNA extract, wherein determining the editing profile of the PDE8A pre-mRNA comprises detecting the ned (non edited isoform) and B isoforms.

2. The method according to claim 1 , further comprising measuring the expression of said editing enzymes ADAR1a, ADAR1b and ADAR2 in the subject sample.

3. The method according to claim 1 , further comprising measuring the expression of mRNAs encoding said editing enzymes ADAR1a, ADAR1b and ADAR2 in the cellular RNA extract.

4. The method according to claim 2 , wherein the expression of said editing enzymes ADAR1a, ADAR1b and ADAR2 is measured by measuring expression of mRNAs encoding said editing enzymes or by measuring expression of said editing enzyme proteins.

5. The method according to claim 2 , wherein expression of said editing enzymes ADAR1a, ADAR1b and ADAR2 in the subject sample is measured quantitatively.

6. The method according to claim 1 , wherein the biological sample is a blood sample comprising white cells.

7. The method according to claim 1 , wherein the step c) of determining the editing profile of the PDE8A pre-mRNA further comprises detecting an isoform selected from I, J, K, L, M and N.

8. The method according to claim 1 , wherein the step c) of determining the editing profile of the PDE8A pre-mRNA further comprises detecting an isoform selected from AB, ABC, ABE, ABEF, ABEFG, ABG, BC, BD, BE, BEG, BF, BFG, BG, and M.

9. The method according to claim 1 , wherein the step c) of determining the editing profile of the PDE8A pre-mRNA comprises detecting the ned, B and AB isoforms.

10. The method according to claim 9 , wherein the step c) of determining the editing profile of the PDE8A pre-mRNA comprises detecting the ned, B, AB and BC isoforms.

11. The method according to claim 1 , wherein the editing profile of the PDE8A pre-mRNA is determined by a process comprising performing a reverse transcription reaction on the cellular RNA extract and performing a nested type PCR comprising two rounds of PCR on the product of the reverse transcription, and wherein: a) the first round of PCR is carried out by the following sets of primers: [table included] and wherein b) the second round of PCR is carried out by the following set of primers: [table included]

12. The method according to claim 1 , wherein ADARs specific isoforms are determined in the method, and wherein the pair of primers specific for the human ADAR mRNA PCR amplification are selected from the group consisting of: (A) for ADAR1-150 isoform m RNA amplification: [table included] (B) for ADAR1-110 isoform m RNA amplification: [table included] (C) for ADAR2 mRNA amplification: [table included]

13. The method of claim 1 wherein the subject is a depressed patient or a suicide attempter.

14. The method of claim 1 wherein the editing profile of the PDE8A pre-mRNA in said cellular RNA extract matches the editing profile of the PDE8A pre-mRNA in reference patients.

15. The method of claim 1 wherein the editing profile of the PDE8A pre-mRNA in said cellular RNA extract matches the editing profile of the PDE8A pre-mRNA for a patient diagnosed with a pathology selected from the group consisting of psychiatric disorders, mental disorders, schizophrenia, depression, Bipolar disease, suicide, abnormal feeding behaviour, Mild Cognitive Impairement (MCI), Epilepsia, Alzheimer and Chronical pain syndromes.

16. The method of claim 1 wherein the editing profile of the PDE8A pre-mRNA in said cellular RNA extract matches the editing profile of the PDE8A pre-mRNA for a patient undergoing treatment for a pathology selected from the group consisting of psychiatric disorders, mental disorders, schizophrenia, depression, Bipolar disease, suicide, abnormal feeding behaviour, Mild Cognitive Impairement (MCI), Epilepsia, Alzheimer and Chronical pain syndromes.

WO 2008/152146 December 2008
WO 2010/070074 June 2010

Lowe et al., Nucleic Acid Research 18(7), 1757-61 (1990). cited by applicant
Robert J. Orlowski et al., “Altered editing in cyclic nucleotide phosphodiesterase 8A1 gene transcripts of systemic lupus erythematosus T lymphocytes”, Immunology vol. 125, pp. 408-419 (2008). cited by applicant
Peng Wang et al., “Human phosphodiesterase 8A splice variants: cloning, gene organization, and tissue distribution”, Gene vol. 280, pp. 183-194 (2001). cited by applicant
S. Dracheva et al., “Increased serotonin 2C receptor mRNA editing: a possible risk factor for suicide”, Molecular Psychiatry, vol. 13, pp. 1001-1010 (2008). cited by applicant
Peter Holmans et al., “Genomewide Significant Linkage to Recurrent, Early-Onset Major Depressive Disorder on Chromosome 15q”, Am. J. Hum. Genet., vol. 74, pp. 1154-1167 (2004). cited by applicant
Peter Holmans et al., Genetics of Recurrent Early Onset Major Depression (GenRED): Final Genome Scan Report, vol. 164, pp. 248-258 (2007). cited by applicant
Eva J. Riedmann et al., “Specificity of ADAR-mediated RNA editing in newly identified targets”, RNA, vol. 14, pp. 1110-1118 (2008). cited by applicant
Takuto Hideyama et al., “Novel Etiological and Therapeutic Strategies for Neurodiseases: RNA Editing Enzyme Abnormality in Sporadic Amyotrophic Lateral Sclerosis”, Journal of Pharmacological Sciences, vol. 113, pp. 9-13 (2010). cited by applicant
Noriyuki Suzuki et al., Ten Novel Mutations of the ADAR1 Gene in Japanese Patients with Dyschromatosis Symmetrica Hereditaria, The Society for Investigative Dermatology, vol. 127, pp. 309-311 (2007). cited by applicant
Harry Towbin et al., “Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications”, Proc. Natl. Acad. Sci. USA, vol. 76, No. 9. pp. 4350-4354 (1979). cited by applicant
Jin Billy Li et al., “Genome-Wide Identification of Human RNA Editing Sites by Parallel DNA Capturing and Sequencing”, Science, vol. 324, pp. 1210-1213 (2009). cited by applicant
Atheir I. Abbas et al., “Assessing serotonin receptor mRNA editing frequency by a novel ultra high-throughput, sequencing method”, Nucleic Acids Research, vol. 38, No. 10, pp. 1-13 (2010). cited by applicant
Michael V. Morabito et al., “High-Throughput Multiplexed Transcript Analysis Yields Enhanced Resolution of 5-Hydroxytryptamine2C Receptor mRNA Editing Profiles”, Molecular Pharmacology, vol. 77, No. 6, pp. 895-902 (2010). cited by applicant
Nader Pourmand et al., “Direct electrical detection of DNA synthesis”, Proc Natl. Acad. Sci., vol. 103, No. 17, pp. 6466-6470 (2006). cited by applicant
Erik P. Anderson et al., “A System for Multiplexed Direct Electrical Detection of DNA Synthesis”, Sens Actuators B Chem., vol. 129, pp. 79-86 (2008). cited by applicant
Tim D. Werry et al.. “RNA editing of the serotonin 5HT2C receptor and its effects on cell signaling, pharmacology and brain function”, Pharmacology & Therapeutics, vol. 119, pp. 7-23 (2008). cited by applicant
Peter H. Seeburg et al., “RNA editing of brain glutamate receptor channels: mechanism and physiology”, Brain Res. Reviews, vol. 26, pp. 217-229 (1998). cited by applicant
Daniel P. Morse et al., “RNA hairpins in noncoding regions of human brain and Caenorhabditis elegans mRNA are edited by adenosine deaminases that act on RNA”; Proc. Natl Acad. Sci. USA, 7906-7911 (2002). cited by applicant
M. Ohman; “A-to-I editing challenger or ally to the microRNA process”, Biochimie, vol. 89, pp. 1171-1176 (2007). cited by applicant
Peter McGuffin et al., “Whole genome linkage scan of recurrent depressive disorder from the depression network study”, Human Molecuiar Genetics, vol. 14, pp. 3337-3345 (2005). cited by applicant
Yu Feng et al., “Association of the Neurotrophic Tyrosine Kinase Receptor 3 (NTRK3) Gene and Childhood-Onset Mood Disorders”, American Journal of Psychiatry, vol. 154, pp. 610-616 (2008). cited by applicant
Ranjana Verma et al., “Linkage Disequilibrium Mapping of a Chromosome 15q25-26 Major Depression Linkage Region and Sequencing of NTRK3”, Biol. Psychiatry, vol. 63, pp. 1185-1189 (2008). cited by applicant
Charles L. Raison. M.D. et al., “Activation of CNS Inflammatory Pathways by Interferon-alpha: Relationship to Monoamines and Depression”; Biol. Psychiatry, vol. 65, pp. 296-303 (2009). cited by applicant
Weidong Yang et al., “Altered RNA editing of serotonin 5-HT2C receptor induced by interferon: Implication for depression associated with cytokine therapy”. Molecular Brain Research, vol. 124, pp. 70-78 (2004). cited by applicant
Alain Poyau et al., “Identification and relative quantification of adenosine to inosine editing in serotonin 2c receptor mRNA by CE”, Electrophoresis, vol. 28, pp. 2843-2852 (2007). cited by applicant
L. Cavarec et al., 'In vitro screening for drug-induced depression and/or suicidal adverse effects: a new toxicogencmic assay based on CE-SSCP analysis of HTR2C mRNA editing in SH-SY5Y cells, Neurotox Res., pp. 49-62 (2013) (Abstract). cited by applicant

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