Utility of Branched DNA Hybridization Methodology for the Quantitation of Oligonucleotides

Transcription

1 Utility of Branched DNA Hybridization Methodology for the Quantitation of Oligonucleotides Laboratory Sciences, MPI Research, A Charles River Company Amy Smith, BA, Senior Director, Bioanalytical/Analytical Services EBF Focus Workshop May 14-15

2 Presentation Outline Emergence of Oligonucleotide Therapeutics Comparison of Assay Platforms Assay Platform Descriptions Branched DNA Assay Details Comparison Data Set: Case Study Topics for Consideration 2

3 Emergence of Oligonucleotide Therapeutics Oligonucleotides bring the hope of a new class of therapeutics Potential treatment for a variety of medical conditions, including difficult to target autoimmune diseases Can target mechanisms of action by interfering with or silencing protein translation Able to enhance or inhibit protein translation Minimal side effects when compared to other therapeutic classes Synthesis and commercial scale up is more feasible when compared to cell or other biologic therapeutics

4 Emergence of Oligonucleotide Therapeutics Approved Oligonucleotide Therapies Mechanism and Indication 1998 Formivirsen Antisense (AON) antiviral drug that was used in the treatment of cytomegalovirus retinitis in immunocompromised patients 2004 Pegaptanib Antisense therapy used for Neovascular (wet) age-related macular degeneration (AMD) 2013 Mipomersen Antisense therapy used for Homozygous familial hypercholesterolemia 2016 Nusinersen Antisense therapy for Spinal Muscular Atrophy (SMA) 2016 Eteplirsen Antisense therapy for the treatment of Duchenne muscular dystrophy (DMD)

5 Oligonucleotide Therapeutics How They Work Antisense Mechanism Hybridize oligos and inactivate mrna to inhibit protein translation through RNAse H-mediated degradation of target RNA or non-degradative steric block of target RNA typically short (8-50 nucleotides), synthetic, single-stranded oligos Chemically modified for stability and target specificity Primary mechanism currently used for marketed oligos Examples: small interfering RNA (sirna), micro RNA (mirs), Gapmer AON

6 Oligonucleotide Therapeutics How They Work Modified mrna technology Delivery of mrna directly to the cell resulting in protein translation Delivery to the cytoplasm for protein translation Or target delivery to specialized cellular components Primary delivery mechanism through lipid nanoparticles or AAV vectors mrna does not need to enter the nucleus or integrate into the genome

7 Oligonucleotide Platform Comparison qrt-pcr Assay Pros Industry standard for oligo measurement Sensitivity Less specific targets are needed Cons Extensive sample cleanup Loss of signal during conversion to cdna Polymerase activity is rate limiting Contamination affecting back ground signal Decreased selectivity based on the use of smaller primers dd-pcr Assay Pros Higher through put when compared to qrt-pcr Increased sensitivity compared to qrt-pcr Less specific targets are needed Cons Extensive sample cleanup Loss of signal during conversion to cdna Contamination affecting back ground signal Selectivity based on the use of smaller primers bdna Assay Pros Sample clean up is not required; matrix is cell lysate No conversion to cdna; eliminate potential loss High throughput using 96- well plate and ELISA-like workflows Enhanced selectivity due to longer base pairs for probes; designer probes Multi-plexing options Cons Currently a single source of probes Patent issues in sharing mrna sequence (including transporter sequences) to develop label probes 7

8 Oligonucleotide Platform Comparison qrt-pcr Assay Pros Industry standard for oligo measurement Sensitivity Less specific targets are needed Cons Extensive sample cleanup Loss of signal during conversion to cdna Polymerase activity is rate limiting Contamination affecting back ground signal Decreased selectivity based on the use of smaller primers dd-pcr Assay Pros Higher through-put when compared to qrt-pcr Increased sensitivity compared to qrt-pcr Less specific targets are needed Cons Extensive sample cleanup Loss of signal during conversion to cdna Polymerase activity is rate limiting Contamination affecting back ground signal Selectivity based on the use of smaller primers bdna Assay Pros Sample clean up is not required; matrix is cell lysate No conversion to cdna; eliminate potential loss High throughput using 96- well plate and ELISA-like workflows Enhanced selectivity due to longer base pairs for probes; designer probes Multi-plexing options Cons Currently a single source of probes Patent issues in sharing mrna sequence (including transporter sequences) to develop label probes 8

9 Oligonucleotide Platform Comparison qrt-pcr Assay Pros Industry standard for oligo measurement Sensitivity Less specific targets are needed Cons Extensive sample cleanup Loss of signal during conversion to cdna Contamination affecting back ground signal Decreased selectivity based on the use of smaller primers Single target analysis only dd-pcr Assay Pros Higher through-put when compared to qrt-pcr Increased sensitivity compared to qrt-pcr Less specific targets are needed Cons Extensive sample cleanup Loss of signal during conversion to cdna Polymerase activity is rate limiting Contamination affecting back ground signal Selectivity based on the use of smaller primers Single target analysis bdna Assay Pros Sample clean up is not required; matrix is cell lysate No conversion to cdna; eliminate potential loss High throughput using 96- well plate and ELISA-like workflows Enhanced selectivity due to longer base pairs for probes; designer probes Multi-plexing options Cons Currently a single source of probes Patent issues in sharing mrna sequence (including transporter sequences) to develop label probes Hybridization takes hours 9

10 qrt-pcr for RNA Measurement One-step vs. Two-step qrt-pcr One-step assays combine reverse transcription and PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase. One-step qrt-pcr only utilizes sequence-specific primers. Two-step assays, the reverse transcription and PCR steps are performed in separate tubes, with different optimized buffers, reaction conditions, and priming strategies. Quantitative reverse transcription polymerase chain reaction (qrt-pcr) 1. Isolation and purification of the RNA from the sample 2. Transcription of purified RNA into complementary DNA (cdna) by reverse transcriptase from total RNA or messenger RNA (mrna) 3. Sequence specific primers bind to cdna 4. The cdna is then used as the template for the qpcr reaction 5. Detection mechanism is via non-specific fluorescent dyes or sequence specific oligo fluorescent probes

11 ddpcr for RNA Measurement Droplet Digital PCR (ddpcr) 1. Isolation and purification of the RNA from the sample 2. Transcription of purified RNA into complementary DNA (cdna) by reverse transcriptase from total RNA or messenger RNA (mrna) 3. The cdna is then used as the template for the ddpcr reaction 4. ddpcr fractionates target DNA into ~20,000 droplets 5. Droplets undergo amplification 6. Detection via fluorescence

12 bdna for mrna Measurement Branched DNA (bdna) Hybridization based assay using the inherent characteristics of RNA to add sequence specific probes and labels to directly measure RNA present in a sample lysate. Assay can be done using 96-well plates and ELISA-based workflows, facilitating high throughput analysis.

13 bdna Workflow Target Capture RNA Capture Target Probe Set contains specific probes to target ~400 nucleotide region of target Capture Extender (CE) composed of generic and target-specific regions Each capture extender binds unique region of target RNA Designed for multiple binding sites for robust capture of RNA (cooperative hybridization for improved specificity Label Extender (LE) target (20-25-mer) and pre-amplifier regions Bind next to each other creating a specific template for the preamplifier to bind Blocking Probes (BP) complimentary to target sequence not bound by capture extender or label extender Stabilizes mrna not bound by capture extender or label extender Minimizes non-specific binding which would result in the loss of selectivity 13

14 bdna Workflow Signal Amplification and Detection Signal Amplification Pre-Amplifier and Amplifier DNA create branched DNA structure Label Probe consists of short oligonucleotide bound to alkaline phosphatase bdna structure allows for dense decoration of DNA with Label Probe Each Amplifier contains hybridization sites for 400 Label Probes Density of LE s determine assay sensitivity Signal Detection Alkaline phosphatase cleaves luminescent substrate Luminescent signal is relative to the amount of target mrna in the sample 14

15 Comparison of Assay Platforms Case Study Experimental Design: Platform comparison for hepo mrna quantitation across two laboratory sites 5 C57BL/6 mice dosed with hepo mrna Serum samples taken from each mouse at 1 hour and 24 hours post-dose Samples were then processed using either bdna or PCR (qrt-pcr and/or ddpcr) at two laboratory sites bdna samples assayed via serum lysate preparation PCR (qrt-pcr and/or ddpcr) via mrna isolation and purification followed by subsequent transcription to cdna 15

16 Comparison of Assay Platforms Case Study Quantification of hepo mrna by site and platform 16

17 Comparison of Assay Platforms Case Study: Part A Single Site Comparison of bdna, qrt-pcr and ddpcr for hepo mrna 1 hr post dose 24 hr post dose Observations: Comparable data observed for qrt-pcr and ddpcr qrt-pcr and ddpcr data was generated from samples isolated and purified on the same day, by the same analyst Experimental variables through cdna generation were controlled Large magnitude of difference observed when comparing data generated by bdna assay platform against traditional PCR platforms Observation led to another question, was the bdna data real? 17

18 Comparison of Assay Platforms Case Study: Part A (continued) Single Site Comparison of qrt-pcr and ddpcr for hepo mrna Observations: Comparable data observed between qrt-pcr and ddpcr platforms Same isolation and purification process was used for both platforms Would differences have been observed if the isolation and purification had been different? Is assay variability primarily in the isolation and purification steps? 18

19 Comparison of Assay Platforms Case Study: Part B Multi-site Comparison of bdna for hepo mrna Multi-site Comparison of qrt-pcr for hepo mrna Observations: Comparable data for bdna platform across sites Variability in qrt-pcr between sites; variables controlled in previous experiment for comparison of ddpcr and qrt-pcr were not controlled (i.e., site, analyst and isolation/purification processing) 19

20 Comparison of Assay Platforms Case Study Correlation of bdna assay results with qrt-pcr assay results b D N A ( n g / m l ) R 2 = q P C R ( n g / m l ) 20

21 Case Study: Conclusions qrt-pcr and ddpcr platforms may result in an underestimation of therapeutic exposure Loss of mrna signal using PCR assays could be a result of: Nonselective RNA purification during sample preparation Not reaching saturation levels of reverse transcriptase during enzymatic reaction Rate limiting polymerase activity RT-PCR and ddpcr assay platforms generated consistent results Analyst to analyst variability observed for qrt-pcr bdna assay platform out performed traditional PCR assay platforms Indicates higher therapeutic exposure; potentially due to improved assay selectivity Less site to site variability Workflows consistent with ELISA based assays; providing opportunity for high throughput analysis Ability to introduce automation to workflow 21

22 Topics for Consideration Challenges in Bioanalysis with the increase in oligonucleotide therapeutics: Platform selection: Is it time to re-evaluate the gold standard for assay selection? Regulatory requirements for exposure data: Do platforms like bdna fall under the BMV? Should they? What should assay acceptance criteria be? What are the validation criteria? Is duplicate analysis required? Is ISR required? 22

23 Acknowledgments Charles River Labs Jessica St. Charles, Ph.D. Kristin Coulter, Ph.D. Mark Wolfe Michael Helmus Michael Stone Roger Hayes, Ph.D. Affymetrix Moderna Alex Bulychev, Ph.D. Meredith Wolfram Tiffany Palmer 23