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  • Quantitation of Oligonucleotides Using a Hybridization Method in Various Tissues and Species

    for Accurate Pharmacology and Toxicology Evaluations DANIELLE SALHA


  • Oligonucleotides (ONs) and their ApplicationsOligonucleotides (ONs) and their Applications

    Hybridization-Based Assays to Quantitate ONsHybridization-Based Assays to Quantitate ONs

    Quantitations of ONs in TissuesQuantitations of ONs in Tissues

    Challenges and Solutions when Quantitating ONs in TissuesChallenges and Solutions when Quantitating ONs in Tissues


    Take Home MessagesTake Home Messages


  • Oligonucleotides (ONs)

    Usually consist of 15 to 20 nucleotides (complementary to target mRNA sequence) Unmodified oligonucleotides are rapidly degraded by nucleases, and RNA transcripts do not easily cross biological membranes Chemically modified ribonucleotides are used to protect against nuclease degradation, improve target affinity and delivery to the intended target/tissue/region Covalent attachment of ON to various ligands are designed to improve biodistribution and cellular uptake, or targeting of specific tissues – Peptides, proteins, carbohydrates, aptamers, and small molecules;

    including cholesterol, tocopherol or folic acid

  • ONs — Past and Present

    Lundin K.E. et al. 2015

  • ONs — Broad Range of Applications

    Antisense oligonucleotides (ASOs) – Inhibitors of RNA activity: siRNAs, miRNA mimics, splicing modulators and RNAseH-

    dependent ASOs Modulators of protein activity (Aptamers) mRNAs encoding for therapeutic proteins or vaccine agents Immunostimulatory oligonucleotides Genetic information-reprogramming nucleic acids (CRISPR)

    siRNA: small interfering RNA

    miRNA: micro RNA

    CRISPR: Clustered Regulatory Interspaced Short Palindromic Repeats

  • Four Different Mechanisms of Action for ONs

    2016, Mariana Lagos Quintana Master of Drug Regulatory Affairs

    RISC: RNA induced silencing complex

  • Regulatory Considerations

    Most ONs are evaluated as synthetically manufactured drugs, and follow ICH S2A and S2B guidelines Non-clinical development and safety evaluations of ON therapeutics have generally followed the regulatory guidelines for small molecules There are no ICH or FDA regulatory guidelines that specifically address the quality expectations/standards for oligonucleotide products ON diversity with different mechanisms of action can cause diverse toxicology profiles/concerns – ON-based therapeutic toxicities can be due to interactions between the ON molecule and

    proteins as a result of Watson and Crick base-pairing to unintended nucleic acids or through an independent mechanism.

  • Distribution of ONs in Tissues and Cellular Uptake

    Following different routes of administration, ONs are found at highest concentrations in the liver and kidneys; the spleen follows as the tissue with highest concentration ONs do not cross the blood-brain barrier to target the central nervous system – Therapeutic ONs need to be administered intrathecally — some successes reported Therapeutic ONs are mostly taken up through endocytosis, indicating that the ON must leave the endosome to reach its target in the cytosol or the nucleus – Thus, to improve ON design leading to more efficient drugs, it is important to consider all

    endocytosis routes and trafficking mechanisms in the target cells

  • Immunogenicity and Safety Assessments of ONs

    As ONs are also covalently linked to various ligands, immunogenicity to the linker and ONs can be enhanced With ONs having different functions and applications, their immunogenicity characteristics are likely to be different Very limited publications on anti-RNA antibodies ASO can bind to proteins, such as human serum albumin, which can serve as a carrier to induce immune responses to the ASO

    ASO: Antisense Oligonucleotide

  • Hybridization-Based Assays to Quantitating ONs

  • Hybridization-Based Assays (ELISA)

    Method specific to the ON – Sequence-specific (complementary nucleic acid probes: capture/detection) Widely used in the industry Bioanalytical methods support preclinical and clinical programs – Toxicokinetic (TK)/pharmacokinetic (PK) distribution and elimination studies Validated assays in accordance with FDA guidance1 and Crystal City 2,3 recommendations

    1. FDA. Guidance for Industry: Bioanalytical Method Validation. Rockville, MD:US Department of Health and Human Services, FDA, CDER; 2001.

    2. DeSilva B. et al. (2003) Pharm. Res. 20 (3) 1885-1900

    3. Viswanathan CT et al. (2007) AAPS J. 2007;9: Article 4.

  • Variety of Hybridization Assay Formats for Bioanalysis

    There are three hybridization ELISA formats: Dual-hybridization ELISA Ligation-hybridization ELISA (patent protected) Nuclease-dependent hybridization ELISA (patent protected)

  • Dual Hybridization ELISA Oligonucleotide Bioanalytical Services

    Detection probe

    Capture probe

    Single-Stranded Oligonucleotide

    Enhanced sensitivity over Ligation hybridization ELISA

    Wider dynamic range

    Disadvantage: Cross-hybridization with 3’ and 5’ metabolites

    Efler, SM et al.(2005) Oligonucleotides,.15 (2) 119–131

  • Ligation Hybridization ELISA Oligonucleotide Bioanalytical Services

    Ligation to test ON with T4 DNA ligase/ATP

    Non-Ligated probe will be washed away

    Advantage: Minimal 3’-metabolite cross- reactivity

    Detection probe

    Single-Stranded Oligonucleotide Capture probe

    Yu RZ et al.(2002) Anal. Biochem. 304, 19-25

    Capture probe: complementary nucleic acid sequence to the ON to capture the ON

    Detection probe: complementary nucleic acid sequence to the ON for detection through the label

  • Nuclease Dependent Hybridization ELISA Oligonucleotide Bioanalytical Services

    The S1 nuclease is a single-strand-specific enzyme cleaving all free capture probe and imperfectly formed duplexes.

    Advantage: Only full-length test ON remains intact and is quantitated

    Wei X et al.(2006) Pharmaceutical Research, Vol 23, No.6

    Capture and

    detection probe

    Single-Stranded Oligonucleotide

  • Hybridization ELISA can be Used with Different Tissues and Matrices to Quantitate ONs



    Brain Liver Eyes Gut

    Spinal Cord Kidney Feces

    Plasma CSF

  • Other Bioanalytical Tools Compared to Hybridization AssayMethod Advantages Limitations

    Capillary Gel Electrophoresis (CGE)

    High resolution for parent and metabolites Need of internal standard

    Widely used to support pre clinical and clinical studies

    Elaborate pre-extraction procedure

    The exact nature of size-separated peaks cannot always be determined

    Extensive sample clean up Low sensitivity

    Quantitative PCR Highest sensitivity Extensive optimization of the method is required

    Little to no sample processing required Lacks precision and accuracy

    No resolution for metabolite quantification

    High Resolution MS Improved sensitivity compared to CGE Time-consuming

    Good resolution of parent and metabolites Extract-dependent and complex spectra

    Hybridization ELISA

    No sample clean up (plasma) or minimum sample cleanup (Tissue)

    Narrower calibration range than chromatographic methods (10- to 50-fold)Low reagents cost

    Very high sensitivity, precision and accuracy

    Highly selective of parent Quantitation of parent / total detectable oligonucleotide metabolites

    (shortmers) not quantifiable in parent assay Widely used to support pre clinical and clinical studies

    High target specificity

  • High Sensitivity Oligonucleotide Bioanalytical Services

    Hybridization ELISA

    Double Ligation

    Capture/Detection Probes DNA Template Probe

    0.2500 ng/mL 1.00 ng/mL

    50.00 ng/g 50.00 to 62.50 ng/g

    LLOQ (Plasma)

    LLOQ (Tissues)


    3.00 to 100.00 ng/mL

    300.00 to 12000 ng/g

    CGE-UV: Capillary gel electrophoresis of single-stranded DNA fragments with UV detection

  • Other Bioanalytical Tools Compared to Hybridization Assay

    Method Sensitivity Selectivityfor Parent

    Resolution of Parent and Metabolites

    limited Sample Clean up Specificity

    Accuracy and


    Wide Dynamic Range

    Total Top Performance

    Capillary Gel Electrophoresis

    (CGE) + + ++++ + ++ ++++ ++++ 3

    Quantitative PCR +++++ + + ++++ ++++ + ++ 3

    High Resolution MS ++ + ++++ + ++ ++++ ++++ 3

    Hybridization ELISA ++++ ++++ + ++++ ++++ ++++ ++ 5

  • Time Associated with Hybridization Assays

    Method development: 12 -15 days for plasma and 5 extra days for tissue Validation: 10-15 days for validation Sample analysis: 60-90 samples per day Turnaround: 2 weeks for first-in-human studies (sample analysis, QC and QA’ed data transferred to sponsor)

  • Method Validation

    Goal: Prove the reliability, robustness and reproducibility of the assay – Acceptance criteria for validation parameters predefined – Generally 20-25% for ligand-binding assays QC samples prepared in the same biological m


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