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DRACO

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DRACO

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  1. DRACO ? (Double-stranded RNA Activated Caspase Oligomerizer) ? Broad-Spectrum Antiviral Therapeutics ? RIDER Institute Revolutionary Innovation, Development, Education, and Research Dr. Todd H. Rider? thor@riderinstitute.org? A mighty creature is the germ,? Though smaller than the pachyderm.? His customary dwelling place? Is deep within the human race.? His childish pride he often pleases? By giving people strange diseases.? Do you, my poppet, feel infirm?? You probably contain a germ.? THR-1 2016-01-12 ?-Ogden Nash?

  2. DRACO Broad-Spectrum Antiviral Therapeutics ? Dr. Todd H. Rider ?thor@riderinstitute.org ? Nontoxic & effective in 13 cell types:? •  Monkey Vero E6 kidney cells? •  Normal human lung fibroblasts? •  Normal human hepatocytes? •  Normal human airway epithelial cells? •  Normal human osteoblasts? •  Normal human aortic muscle cells? •  Human embryonic kidney 293 cells? •  Human HeLa cells? •  Pig ST cells? •  Pig SK-RST cells? •  Mouse L929 cells? •  Mouse BALB/3T3 cells? •  Mouse NIH/3T3 cells? Live mice—nontoxic, effective against:? •  Influenza H1N1 A/PR/8/34? •  Amapari arenavirus? •  Tacaribe arenavirus? •  Guama bunyavirus? Effective against 18 viruses:? •  Dengue type 2 flavivirus? •  Amapari arenavirus? •  Tacaribe arenavirus? •  Guama Be An 277 bunyavirus? •  Guama Be Ar 12590 bunyavirus? •  Influenza H1N1 A/PR/8/34? •  Influenza H1N1 A/WS/33? •  Adenovirus 5? •  Murine adenovirus? •  Reovirus 3? •  Theiler’s encephalomyelitis? •  Rhinovirus 1B? •  Rhinovirus 2? •  Rhinovirus 14? •  Rhinovirus 30? •  Coronavirus TGEV Purdue? •  Coronavirus TGEV Miller? •  Coronavirus TGEV AR310? THR-2 2016-01-12

  3. DRACO: Scientific and Public Reception ?   First published in? PLoS ONE (2011)? DRACO has been covered by:? U.S. News & World Report? Wall Street Journal? Bloomberg Business Week? Technology Review? New Scientist? Popular Science? National Geographic? R&D Magazine? International Business Times? Nature Biotechnology? Boston Globe? Wired? BBC? NPR? Discovery Channel? Etc.?   Presented at numerous? international conferences?   Protected by U.S. and? international patents? and patent applications? One of Time magazine’s? top inventions of the year? (Nov. 28, 2011, pp. 58, 78)? Called “visionary”? by the White House? (April 2012, p. 9)? Cover story in? Science Illustrated? (September 2012)? Featured on BBC? Horizon TV program? (May 2013)? THR-3 2016-01-12

  4. Only a Few Antiviral Treatments Exist Now, And Those Have Major Disadvantages Drugs that target components? used by specific viruses? Amantadine? Saquinavir? Vaccines? RNAi? Influenza A? M2 channel? HIV? Specific? pathogen gene? Louis Pasteur? preparing? rabies vaccine? protease? •  Time-consuming and expensive to create? drugs for each virus? •  Drugs are so specific that resistant virus? strains can easily arise naturally or artificially? •  Some drugs can have serious adverse effects? General immune system boosters? •  Separate vaccines must be produced? for each virus, or even each virus strain? •  Substances such as interferon? have been tested since the 1950s? •  It is extremely difficult to create? vaccines for some viruses? •  This approach is ineffective? against many pathogens? •  To be effective, some vaccines must? be administered before infection ? •  Potential adverse effects? include flu-like symptoms,? autoimmune disease,? shock, and death? Interferon-α? •  Viruses may be designed to evade? existing vaccines? THR-4 2016-01-12

  5. Double-stranded RNA Activated Caspase Oligomerizer (DRACO)
 Selectively Kills Virus-Infected Cells ? DRACOs? DRACOs? do not harm? healthy cells? Healthy cell? Infected? cell with? viral dsRNA? Infection? terminated? Healthy cell? THR-5 2016-01-12

  6. Double-stranded RNA Activated Caspase Oligomerizer (DRACO)
 Selectively Kills Virus-Infected Cells ? Viral dsRNA? Apoptosis? (cell suicide)? induction? domain? (Apaf-1 1-97)? dsRBM #2? dsRBM #1? RNA and DNA? viruses produce? long double-? stranded RNA? (dsRNA),? but uninfected? mammalian? cells do not? dsRNA binding domain? (PKR 1-181)? DRACO? THR-6 2016-01-12

  7. Double-stranded RNA Activated Caspase Oligomerizer (DRACO)
 Selectively Kills Virus-Infected Cells ? Viral dsRNA? dsRBM #2? Cellular? pro-caspase 9? Apoptosis? induction? domain? dsRBM #1? dsRNA? binding? domain? dsRBM #2? dsRBM #1? THR-7 2016-01-12

  8. Double-stranded RNA Activated Caspase Oligomerizer (DRACO)
 Selectively Kills Virus-Infected Cells ? Viral dsRNA? Crosslinking? cellular? pro-caspase 9? rapidly kills? virus-infected cell? dsRBM #2? Apoptosis? induction? domain? dsRBM #1? dsRNA? binding? domain? dsRBM #2? dsRBM #1? DRACO should be effective against? a broad spectrum of viruses? DRACO should not harm uninfected cells? THR-8 2016-01-12

  9. Use Protein Transduction Tags
 To Deliver DRACOs in Vitro and in Vivo ? We have produced DRACOs? with protein delivery tags ? Other researchers showed that tags deliver? any protein to all cell types in live mice ? Protein delivery tags? TAT YGRKKRRQRRR? PTD-4 YARAAARQARA? ARG RRRRRRRRR? Untagged β β-gal protein? cannot get into cells? β β-gal with TAT tag? freely enters cells? ATG-His6 - TAT - DRACO? ATG-His6 - PTD4 - DRACO ? ATG-His6 - ARG - DRACO? pET100? prokaryotic? expression? vector? Transform? E. coli? AmpR? Purify protein? (Ni-NTA column)? DRACOs penetrate? into animal cells? TAT PTD-4 ARG? Ref: S. R.? Schwarze? et al. (1999)? Science? 285, 1569? PKR? Apaf-1? •  Also delivers β β-galactosidase to brain, spleen, etc.? •  No side effects in mice after 2 weeks of use? DRACO? THR-9 2016-01-12

  10. Improving the DRACO Purification Protocol ? THR-10 2016-01-12

  11. DRACO Penetrates into Cells within Minutes ? and Persists inside Cells for Days ? Cells only? Transduction time? Human lung fibroblasts? with no DRACO? DRACO? Marker? 10 min? 15 min? 30 min? 45 min? 1.5 hr? 0 min? 5 min? 1 hr? 2 hr? 3 hr? 4 hr? Molecular weight (kDa)? 53? 36? TAT-DRACO? 28? Cells only? Human lung fibroblasts? + Lumio-labeled DRACO? Marker? Days post-transduction? Molecular weight (kDa)? 0 1 2 3 4? Similar results for other tags? 53? We have also demonstrated? that DRACO is effective when? added up to at least 11 days? before virus, or when any? remaining extracellular DRACO? is removed before adding virus? TAT-DRACO? 36? 28? THR-11 2016-01-12

  12. DRACO Is Effective Against Rhinovirus 1B
 in Normal Human Lung Fibroblasts ? THR-12 2016-01-12

  13. DRACO Is Effective Against Rhinovirus 1B
 in Normal Human Lung Fibroblasts ? No virus Rhinovirus 1B? 100? 75? Cell viability (%)? 50? 25? 0? ARG-PKR1-181? ARG-PKR-Apaf? + ARG-Apaf1-97? ARG-Apaf1-97? Untreated? + PTD-Apaf1-97? PTD-PKR-Apaf? PTD-PKR1-181? PTD-Apaf1-97? + TAT-Apaf1-97? TAT-PKR1-181? TAT-PKR-Apaf? ARG-PKR1-181? TAT-Apaf1-97? PTD-PKR1-181? 50x E. coli? TAT-PKR1-181? extract? DRACO? DRACO? DRACO? THR-13 2016-01-12

  14. DRACO Reduces Rhinovirus Titers 1x105? NHLF supernatant analyzed by TCID50 assays 3x104? 1x104? Day-4 rhinovirus titer (pfu/ml) 3x103? 1x103? 3x102? 1x102? 3x101? 1x101? Below limit of detection (<2 pfu/ml) 3x100? 1x100? 10x E. coli extract PTD-PKR + PTD-Apaf TAT-PKR + TAT-Apaf PTD- DRACO TAT- DRACO Untreated THR-14 2016-01-12

  15. DRACO Is Effective Against Theiler’s
 Encephalomyelitis in Murine L929 Cells ? THR-15 2016-01-12

  16. DRACO Is Effective Before or After Infection ? 125? No virus Murine adenovirus? Cell viability (%) for L929 cells? 100? 75? 50? 25? 0? Untreated DRACO Untreated DRACO Untreated DRACO Untreated DRACO? Treated 24 hours? before infection? after infection? Treated 72 hours? after infection? Treated 24 hours? Treated 48 hours? after infection? Similar results for other viruses tested? THR-16 2016-01-12

  17. DRACO Is Effective Against Dengue Hemorrhagic Fever Virus ? THR-17 2016-01-12

  18. DRACO Is Effective Against Dengue Hemorrhagic Fever Virus ? 125? No virus Dengue? 100? Cell viability (%)? 75? 50? 25? 0? 10x E. coli? DRACO mutant? DRACO mutant? PTD-PKR-Apaf? Untreated? PTD-PKR1-181? PTD-PKR-Apaf? PTD-Apaf1-97? + PTD-Apaf1-97? TAT-PKR-Apaf? + TAT-Apaf1-97? TAT-PKR1-181? TAT-Apaf1-97? PTD-PKR1-181? TAT-PKR-Apaf? TAT-PKR1-181? extract? DRACO? DRACO? THR-18 2016-01-12

  19. Preliminary Data Shows DRACO Reduces
 Titers of Hemorrhagic Fever Virus in Cells ? RT-qPCR Viral Titers of Tacaribe Arenavirus in Vero E6 Cells? 4 Days Post Infection? 8 Days Post Infection? THR-19 2016-01-12

  20. Median Effective Concentration (EC50) ? for DRACO is ~2-3 nM ? Theiler’s encephalomyelitis? Murine adenovirus? Rhinovirus 1B? 100? 75? Cell viability (%)? 50? 25? 0? 0.1 0.3 1 3 10 30 100? DRACO concentration (nM)? THR-20 2016-01-12

  21. DRACO Is Nontoxic in Mice and
 Has Good Pharmacokinetics ? Intraperitoneal administration? (PTD-DRACO)? Intranasal administration? (Lungs)? 1000! 1000! 900! 900! Liver! Kidney! Lung! ARG-DRACO! TAT-DRACO! PTD-DRACO! 800! 800! Concentration ("g DRACO per g tissue)! Concentration ("g DRACO per g tissue)! 700! 700! 600! 600! 500! 500! 400! 400! 300! 300! 200! 200! 100! 100! 0! 0! 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48! 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48! Time after DRACO administration (hours)! Time after DRACO administration (hours)! •  No apparent toxicity in live mice or in tissues examined during necropsy? •  DRACO penetrates into all tissues tested and can persist 24-48 hours? •  Different administration routes and delivery tags are better for different tissues? THR-21 2016-01-12

  22. Intranasal DRACO Greatly Improves Survival, Reduces Weight Loss,
 & Reduces Viral Titers in Mice Challenged with 1 LD50 H1N1 Influenza ? 110.00 110%? Mouse weight relative to Day 0? 100.00 100%? ARG-DRACO (n=8) 90.00 90%? Day-2 lung viral titers reduced 300x by DRACO 80.00 80%? Buffer (n=7) 70.00 70%? Obtained similar results from multiple trials? 60.00 60%? 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13? 1 2 3 4 5 6 7 8 9 10 11 12 13 Days post infection 1 intranasal DRACO dose on day 0? THR-22 2016-01-12

  23. Preliminary Data Shows DRACO Efficacy Against
 Tacaribe Hemorrhagic Fever Virus in AG129 Mice ? Day-9 serum viral titers reduced >30x by DRACO THR-23 2016-01-12

  24. Some “Outbreak” Viruses of Potential Interest ? Family ? Virus ? Human impact ? Alternatives ? BL2 model ? Alpha- ? Now in Americas ? Cells: Yes ? Chikungunya ? No ? virus ? Years of pain ? Mice: Yes ? Junin, Lassa, ? Arena- ? Bioterrorism ? Cells: Yes ? No ? etc. ? virus ? 25% mortality ? Mice: Yes ? Rift Valley ? Bunya- ? Bioterrorism ? Cells: Yes ? No ? Fever, etc. ? virus ? Hemorrhagic fever ? Mice: Yes ? Corona- ? Epidemic? ? Cells: Yes ? MERS ? No ? virus ? 25% mortality ? Mice: Yes ? Filo- ? Bioterrorism ? Some in ? Cells:Partial ? Ebola ? virus ? <90% mortality ? human trials ? Mice: No ? Pox- ? Bioterrorism ? No-engineered ? Cells: Yes ? Smallpox ? virus ? 50% mortality ? resistance ? Mice: Yes ? THR-24 2016-01-12

  25. Some Clinical Viruses of Potential Interest (1 of 2) ? Family ? Virus ? Human impact ? Alternatives ? BL2 model ? Delta- ? Hepatitis ? >20M infected ? HBV vaccine ? Cells:Partial ? virus ? D ? >20,000 deaths/yr ? only ? Woodchuck:Y ? Flavi- ? Hepatitis ? 200M infected ? Side effects, ? Cells: Yes ? virus ? C ? >350,000 deaths/yr ? $$$ ? Mice: Yes ? Flavi- ? Dengue ? 500M/yr infected ? Vaccines in ? Cells: Yes ? virus ? Fever ? ~1M deaths/yr ? development ? Mice: Yes ? Hepadna- ? Hepatitis ? >350M infected ? Vaccine ? Cells: Yes ? virus ? B ? >600,000 deaths/yr ? Treatments ltd ? Mice: Yes ? Hepe- ? Hepatitis ? 20M/yr infected ? Vaccines in ? Cells:Partial ? virus ? E ? >60,000 deaths/yr ? development ? Rabbits: Yes ? THR-25 2016-01-12

  26. Some Clinical Viruses of Potential Interest (2 of 2) ? Family ? Virus ? Human impact ? Alternatives ? BL2 model ? Herpes- ? Varicella ? >3B infected ? Treat but ? Cells: Yes ? virus ? Zoster ? Chronic infection ? not cure ? Mice: Yes ? Herpes- ? Herpes ? >500M infected ? Treat but ? Cells: Yes ? virus ? Simplex ? Chronic infection ? not cure ? Mice: Yes ? Orthomyxo ? 5M/yr infected ? Very ? Cells: Yes ? Influenza ? -virus ? >250,000 deaths/yr ? limited ? Mice: Yes ? Respiratory Paramyxo ? Very serious ? Cells: Yes ? No ? Syncytial ? -virus ? for infants ? Mice: Yes ? Hepatitis ? Picorna- ? >30M infected ? Vaccine ? Cells: Yes ? A ? virus ? >100,000 deaths/yr ? only ? Mice: Yes ? HIV & other ? Retro- ? >35M infected:Health ? Treat not cure ? Cells: Yes ? retroviruses ? viruses ? impact even w drugs ? Resistance ? Mice: Yes ? THR-26 2016-01-12

  27. Potential Work to Optimize DRACOs in Cells
 (Structure-Activity Relationship or SAR Process) ? (2) Evaluate penetration and toxicity? (1) Genetically engineer,? produce, and purify? multiple DRACO versions? of all DRACOs in cells? Mammalian cells? (3) Measure cell viability & viral titers to find? median effective concentration of DRACO? Iterate to optimize? DRACOs? Viruses? Use best DRACOs? from cell experiments? for rodent trials? THR-27 2016-01-12

  28. Potential Work to Optimize DRACOs in Rodents ? Test best DRACOs from cell experiments? in rodent models of virus infection to find:? •  Threshold for toxicity if any? •  Tissue distribution? •  Half-life? •  Metabolized products? •  Immunogenicity? •  Median effective concentration against? virus (morbidity and viral titers)? •  Maximum time pre-infection for? prophylactic DRACO administration? •  Maximum time post-infection for? therapeutic DRACO administration? Iterate DRACO designs to:? •  Minimize toxicity and immunogenicity? •  Maximize distribution and half-life? •  Maximize efficacy against virus? THR-28 2016-01-12

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