Infectious Diseases – Joacim Elmen, Phil Kearney, Sakari Kauppinen, Roche Innovation Center Copenhagen AS
Abstract for “Pharmaceutical composition”
“The invention provides pharmaceutical compositions that contain short single-stranded Oligonucleotides of length between 8 and 17 nucleobases. These are complementary to human microRNAs. These oligonucleotides have a remarkable ability to reduce miRNA repression in vivo. The incorporation of high affinity nucleotide analogs into the oligonucleotides produces highly effective anti-microRNA molecules that appear to work via the formation almost irreversible doubles with the mIRNA targets. This is in contrast to RNA cleavage based mechanisms such as RNaseH and RISC.
Background for “Pharmaceutical composition”
“Definitions”
“Further Embodiments”
“REFERENCES”
“EXPERIMENTAL”
“Example 1 – Monomer Synthesis”
“Example 2: Oligonucleotide Synthesis”
“Abbreviations”
“Example 3 – Design of the LNAAnti-miR Oligonucleotides & Melting Temperatures”
“Example 4 – Stability of LNA Oligonucletides In Human or Rat Plasma”
“Example 5: In Vitro Model: Cell Culture”
“Example 6 – In Vitro Model: Treatment With LNA AntimiR Antisense Oligonucleotide
“Example 7 – In Vitro Model and In Vivo Model: Analysis by Oligonucleotide Inhibition miR Expression using microRNA Specific QuantitativePCR
“Example 8 – Assessment of LNA Antago?Mir Knock?Down Specificity Using miRNA Expression Profiling
“Example 9 – Detection microRNAs using In Situ Hybridization. Detection microRNAs within Formalin-Fixed ParaffinEmbedded Tissue sections by In Situ Hybridization
“Example 10 – In Vitro Model for Analysis and Isolation of mRNA Expression (Total Isolation and Synthesis of mRNA)”
“Example 11 – LNA Oligonucleotide uptake and efficacy in Vivo”
“Example 12: LNA-antimiR-122a Dose-Response In Vivo in C57/BL/J Female Mice”
“Example 12a – Northern Blot”
“Example 13 – Assessment of Cholesterol Levels In Plasma in LNA AntimiR122 Treated Mice
“Example 14 – Assessment of miR-122a Target mRNA levels in LNA-antimiR-122a-treated Mice”
“Example 15 – LNA Oligonucleotide Duration Of Action In Vivo”
“Example 16 – LNA Oligonucleotide Duration Of Action In Vivo”
“Example 17 – Dose dependent miR-122a Target mRNA Induction with SPC3372 Inhibition miR-122a”
“Example 18 – Transient Induction Of miR-122a Target mRNAs After SPC3372 Treatment”
“Example 19 – Induction of VIdIr into Liver by SPC3372 Treatment
“Example 20 – Stability of miR122a/SPC3372 duplex in Mouse Plasma”
“Example 21: Sequestering Mature miR-122a by SPC3372 Leads To Duplex Formation”
“Example 22”: miR-122a Sequestering with SPC3372 and SPC3372 Distribution Assessments by In Situ Hybridization Liver Sections
“Example 23: Micro Array Analysis”
“Example 24. “Example 24. Dose-Dependent MiR-122 Inhibition in Mouse Liver by LNA/antimiR is Enhanced Compared to Antagomir Ihibition miR-122”
“Example 25. “Example 25.
“Example 26 Methods for Performing the LNA-antimiR/Hypercholesterolemic Experiment and Analysis”
“Example 27 Modulation by Hepatitis B Replication by LNA/antimiR (SPC3649).”
“Example 28 Enhanced LNA/antimiR?” Antisense Oligonucleotide Targeting miR-21″
“Example 29 – Luciferase reporter assay for assessing functional inhibition of microRNAs by LNA antimiRs or other microRNA targeting Oligos: Generalisation and Enhancement of New and Enhanced Design as Preferred Design to Block microRNA Function”
“Example 30″: Design of an LNA antimiR library for all Human microRNA sequences in miRBase miRNA Database Version 8.1, Griffiths-Jones (S.), Grocock, R. J. Van Dongen (S.), Bateman, A. and Enright, A. J. 2006. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 34: D140-4 (http://microrna.sanger.ac.uk/sequences/index.shtml)”
Summary for “Pharmaceutical composition”
“Definitions”
“Further Embodiments”
“REFERENCES”
“EXPERIMENTAL”
“Example 1 – Monomer Synthesis”
“Example 2: Oligonucleotide Synthesis”
“Abbreviations”
“Example 3 – Design of the LNAAnti-miR Oligonucleotides & Melting Temperatures”
“Example 4 – Stability of LNA Oligonucletides In Human or Rat Plasma”
“Example 5: In Vitro Model: Cell Culture”
“Example 6 – In Vitro Model: Treatment With LNA AntimiR Antisense Oligonucleotide
“Example 7 – In Vitro Model and In Vivo Model: Analysis by Oligonucleotide Inhibition miR Expression using microRNA Specific QuantitativePCR
“Example 8 – Assessment of LNA Antago?Mir Knock?Down Specificity Using miRNA Expression Profiling
“Example 9 – Detection microRNAs using In Situ Hybridization. Detection microRNAs within Formalin-Fixed ParaffinEmbedded Tissue sections by In Situ Hybridization
“Example 10 – In Vitro Model for Analysis and Isolation of mRNA Expression (Total Isolation and Synthesis of mRNA)”
“Example 11 – LNA Oligonucleotide uptake and efficacy in Vivo”
“Example 12: LNA-antimiR-122a Dose-Response In Vivo in C57/BL/J Female Mice”
“Example 12a – Northern Blot”
“Example 13 – Assessment of Cholesterol Levels In Plasma in LNA AntimiR122 Treated Mice
“Example 14 – Assessment of miR-122a Target mRNA levels in LNA-antimiR-122a-treated Mice”
“Example 15 – LNA Oligonucleotide Duration Of Action In Vivo”
“Example 16 – LNA Oligonucleotide Duration Of Action In Vivo”
“Example 17 – Dose dependent miR-122a Target mRNA Induction with SPC3372 Inhibition miR-122a”
“Example 18 – Transient Induction Of miR-122a Target mRNAs After SPC3372 Treatment”
“Example 19 – Induction of VIdIr into Liver by SPC3372 Treatment
“Example 20 – Stability of miR122a/SPC3372 duplex in Mouse Plasma”
“Example 21: Sequestering Mature miR-122a by SPC3372 Leads To Duplex Formation”
“Example 22”: miR-122a Sequestering with SPC3372 and SPC3372 Distribution Assessments by In Situ Hybridization Liver Sections
“Example 23: Micro Array Analysis”
“Example 24. “Example 24. Dose-Dependent MiR-122 Inhibition in Mouse Liver by LNA/antimiR is Enhanced Compared to Antagomir Ihibition miR-122”
“Example 25. “Example 25.
“Example 26 Methods for Performing the LNA-antimiR/Hypercholesterolemic Experiment and Analysis”
“Example 27 Modulation by Hepatitis B Replication by LNA/antimiR (SPC3649).”
“Example 28 Enhanced LNA/antimiR?” Antisense Oligonucleotide Targeting miR-21″
“Example 29 – Luciferase reporter assay for assessing functional inhibition of microRNAs by LNA antimiRs or other microRNA targeting Oligos: Generalisation and Enhancement of New and Enhanced Design as Preferred Design to Block microRNA Function”
“Example 30″: Design of an LNA antimiR library for all Human microRNA sequences in miRBase miRNA Database Version 8.1, Griffiths-Jones (S.), Grocock, R. J. Van Dongen (S.), Bateman, A. and Enright, A. J. 2006. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 34: D140-4 (http://microrna.sanger.ac.uk/sequences/index.shtml)”
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