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My Long Journey To Antisense Oligos [ 1968 to 1993 ] And The Remaining Journey To

My Long Journey To Antisense Oligos [ 1968 to 1993 ] And The Remaining Journey To Antisense Therapeutics Including Fountain of Youth [ 1993 to 2013 ? ]. James Summerton, Ph.D. (Biochemistry) G ENE T OOLS , LLC. Academic Phase. 1968 Joined Chris Mathews lab in Tucson

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My Long Journey To Antisense Oligos [ 1968 to 1993 ] And The Remaining Journey To

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  1. My Long Journey To Antisense Oligos [ 1968 to 1993 ] And The Remaining Journey To Antisense Therapeutics Including Fountain of Youth [ 1993 to 2013 ? ] James Summerton, Ph.D. (Biochemistry) GENE TOOLS, LLC

  2. Academic Phase 1968 Joined Chris Mathews lab in Tucson Conceived gene-blocking strategy for viruses 1970 1972 Ph.D. in biochemistry: Chris Mathews 1974 Gene-blocking agents begun at Berkeley 1976 Gene-blocking agents continued in Denver 1978 First gene-blocking patent issued (sequence-specific crosslinking) Joined Chris Mathews lab, OSU, Corvallis 1980 Switched to major-groove gene-blocking agents Left OSU, founded first gene-blocking company

  3. Academic Phase 1968 Joined Chris Mathews lab in Tucson Conceived gene-blocking strategy for viruses 1970 1972 Ph.D. in biochemistry: Chris Mathews 1974 Gene-blocking agents begun at Berkeley 1976 Gene-blocking agents continued in Denver 1978 First gene-blocking patent issued (sequence-specific crosslinking) Joined Chris Mathews lab, OSU, Corvallis 1980 Switched to major-groove gene-blocking agents Left OSU, founded first gene-blocking company

  4. First Scheme (1974-1978) Sequence-Specific Crosslinking Agents H Antisense strand (carrier) Activated antisense strand Sense strand (target) Cross-linked carrier-target

  5. Academic Phase 1968 Joined Chris Mathews lab in Tucson Conceived gene-blocking strategy for viruses 1970 1972 Ph.D. in biochemistry: Chris Mathews 1974 Gene-blocking agents begun at Berkeley 1976 Gene-blocking agents continued in Denver 1978 First gene-blocking patent issued (sequence-specific crosslinking) Joined Chris Mathews lab, OSU, Corvallis 1980 Switched to major-groove gene-blocking agents Left OSU, founded first gene-blocking company

  6. Second Scheme (1979-1982) AntiVirals, Inc. Major-Groove Binding Agents Binding GC pair Binding CG pair Binding TA pair Binding AT pair

  7. AntiVirals, Inc. Anti-Genes (1979-1982)

  8. AntiVirals, Inc. Commercial Phase Antisense Oligos (1980-1993) • 1980 Founded first gene-blocking company: ANTIVIRALS, Inc. • 1982 Funding from private investors, NIH (SBIR) • Switched to nucleic-acid-analog gene-blocking agents • 1984 Dupont funding • Devised Morpholino structural type • 1986 • “Won” Oregon lottery • 1988 • 1990 Near-final Morpholino structural type • 1992 Confirmed excellent properties • 1993 Discovered delivery problem

  9. AntiVirals, Inc. Commercial Phase Antisense Oligos (1980-1993) • 1980 Founded first gene-blocking company: ANTIVIRALS, Inc. • 1982 Funding from private investors, NIH (SBIR) • Switched to nucleic-acid-analog gene-blocking agents • 1984 Dupont funding • Devised Morpholino structural type • 1986 • “Won” Oregon lottery • 1988 • 1990 Near-final Morpholino structural type • 1992 Confirmed excellent properties • 1993 Discovered delivery problem

  10. AntiVirals, Inc. Third Scheme (1982-1993) Homopolymer binds Heteropolymer does not bind Heteropolymer binds

  11. Competing structure DNA analog Cheap Good activity

  12. AntiVirals, Inc. Commercial Phase Antisense Oligos (1980-1993) • 1980 Founded first gene-blocking company: ANTIVIRALS, Inc. • 1982 Funding from private investors, NIH (SBIR) • Switched to nucleic-acid-analog gene-blocking agents • 1984 Dupont funding • Devised Morpholino structural type • 1986 • “Won” Oregon lottery • 1988 • 1990 Near-final Morpholino structural type • 1992 Confirmed excellent properties • 1993 Discovered delivery problem

  13. Morpholinos From Ribonucleosides AntiVirals, Inc. Analogs derived from ribonucleosides Binds DNA but not RNA Modeling suggested need for more flexible intersubunit link Best

  14. Morpholinos From Ribonucleosides AntiVirals, Inc. Morpholino phosphorodiamidate structural types = B = A C G U Near-final structure Final structure U T T improves RNA binding affinity T allows sequencing by mass spectrometry

  15. AntiVirals, Inc. Commercial Phase Antisense Oligos (1980-1993) • 1980 Founded first gene-blocking company: ANTIVIRALS, Inc. • 1982 Funding from private investors, NIH (SBIR) • Switched to nucleic-acid-analog gene-blocking agents • 1984 Dupont funding • Devised Morpholino structural type • 1986 • “Won” Oregon lottery • 1988 • 1990 Near-final Morpholino structural type • 1992 Confirmed excellent properties • 1993 Discovered delivery problem

  16. 2’-O-Me RNA siRNA Morph PNA MP S-DNA efficacy - + + + + + + + + + + - + + + + + + + specificity - - + + + + + + + stability - - + + + + + + + + solubility cost of material - - - + + + + + ease of assembly + + - + + + + + + splice modification - - + + + + + + + - - - in vivo delivery - - -

  17. AntiVirals, Inc. Antisense Therapeutics ( 1993 – 1997 ) 1993 Discovered delivery problem Delivery: molecular transport engine (1) 1995 Delivery: scrape delivery (2) 1997 AVI went public Left to start GENE TOOLS, LLC

  18. AntiVirals, Inc. Antisense Therapeutics ( 1993 – 1997 ) 1993 Discovered delivery problem Delivery: molecular transport engine (1) 1995 Delivery: scrape delivery (2) 1997 AVI went public Left to start GENE TOOLS, LLC

  19. The Delivery Challenge Cell entry of oligomeric drugs oligomer drug cytosol pH 7.5 extracellular medium pH 7.4 late endosome pH 5.5 lysosome pH 4.5 degraded oligomer

  20. AntiVirals, Inc. Antisense Therapeutics ( 1993 – 1997 ) 1993 Discovered delivery problem Delivery: molecular transport engine (1) 1995 Delivery: scrape delivery (2) 1997 AVI went public Left to start GENE TOOLS, LLC

  21. Delivery system 1: Molecular Engine AntiVirals, Inc. Molecular Engine For Transporting Drugs Across Cell Membranes James Summerton, Ph.D. ANTIVIRALS, Inc. pH-Mediated Solubility Transition of Weak Acid Moieties EARLY ENDOSOME pH 7 LATE ENDOSOME pH 5 ENDOSOMAL MEMBRANE CYTOSOL pH 7 water soluble water soluble lipid soluble

  22. Delivery system 1: Molecular Engine AntiVirals, Inc.

  23. Delivery system 1: Molecular Engine AntiVirals, Inc. Octanol / Water Partitioning

  24. Delivery system 1: Molecular Engine Molecular Engine: not enough power for Morpholinos so adapted for targeting acidic areas of tumors

  25. AntiVirals, Inc. Antisense Therapeutics ( 1993 – 1997 ) 1993 Discovered delivery problem Delivery: molecular transport engine (1) 1995 Delivery: scrape delivery (2) 1997 AVI went public Left to start GENE TOOLS, LLC

  26. Delivery system 2: Scrape Delivery AntiVirals, Inc. cargo molecules in medium scraped cell adherent cell culture plate

  27. AntiVirals, Inc. Antisense Therapeutics ( 1993 – 1997 ) 1993 Discovered delivery problem Delivery: molecular transport engine (1) 1995 Delivery: scrape delivery (2) 1997 AVI went public Left to start GENE TOOLS, LLC

  28. Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started GENE TOOLS, LLC Delivery: Osmotic delivery (3) 1999 Delivery: Microinjection, egg & early embryo (J Heasman, S Ekker) (4) 2001 Delivery: Special Delivery (5) 2003 Delivery: Endo-Porter (6) 2005 2007 Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) 2011 2013 Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

  29. Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started GENE TOOLS, LLC Delivery: Osmotic delivery (3) 1999 Delivery: Microinjection, egg & early embryo (J Heasman, S Ekker) (4) 2001 Delivery: Special Delivery (5) 2003 Delivery: Endo-Porter (6) 2005 2007 Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) 2011 2013 Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

  30. Delivery system 3: Osmotic Delivery release step wash Morpholino PEG-600 high-osmolarity sugar

  31. Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started GENE TOOLS, LLC Delivery: Osmotic delivery (3) 1999 Delivery: Microinjection, egg & early embryo (J Heasman, S Ekker) (4) 2001 Delivery: Special Delivery (5) 2003 Delivery: Endo-Porter (6) 2005 2007 Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) 2011 2013 Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

  32. Delivery system 4: Microinjection Microinjection Into Eggs & Embryos

  33. Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started GENE TOOLS, LLC Delivery: Osmotic delivery (3) 1999 Delivery: Microinjection, egg & early embryo (J Heasman, S Ekker) (4) 2001 Delivery: Special Delivery (5) 2003 Delivery: Endo-Porter (6) 2005 2007 Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) 2011 2013 Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

  34. Delivery system 5: Special Delivery Morpholino DNA EPEI Relative magnitude Delivery system

  35. Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started GENE TOOLS, LLC Delivery: Osmotic delivery (3) 1999 Delivery: Microinjection, egg & early embryo (J Heasman, S Ekker) (4) 2001 Delivery: Special Delivery (5) 2003 Delivery: Endo-Porter (6) 2005 2007 Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) 2011 2013 Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

  36. Delivery system 6: Endo-Porter Figure 1: Probable Endo-Porter mechanism

  37. Delivery system 6: Endo-Porter

  38. Delivery system 6: Endo-Porter

  39. Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started GENE TOOLS, LLC Delivery: Osmotic delivery (3) 1999 Delivery: Microinjection, egg & early embryo (J Heasman, S Ekker) (4) 2001 Delivery: Special Delivery (5) 2003 Delivery: Endo-Porter (6) 2005 2007 Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) 2011 2013 Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

  40. Delivery system 7: Vivo-Morphlinos

  41. Delivery system 7: Vivo-Morphlinos

  42. Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started GENE TOOLS, LLC Delivery: Osmotic delivery (3) 1999 Delivery: Microinjection, egg & early embryo (J Heasman, S Ekker) (4) 2001 Delivery: Special Delivery (5) 2003 Delivery: Endo-Porter (6) 2005 2007 Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) 2011 2013 Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

  43. Delivery system 8: Receptor/Transport System (in progress) Design objectives cell level high-affinity receptor on nearly all cells efficient endocytosis efficient sorting efficient transport to cytosol organ level efficient transcytosis across blood-brain barrier

  44. Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started GENE TOOLS, LLC Delivery: Osmotic delivery (3) 1999 Delivery: Microinjection, egg & early embryo (J Heasman, S Ekker) (4) 2001 Delivery: Special Delivery (5) 2003 Delivery: Endo-Porter (6) 2005 2007 Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) 2011 2013 Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

  45. Fountain of Youth Hutchinson-Gilford Progeria Syndrome Nuclear membrane: normal Nuclear membrane: HGPS

  46. Fountain of Youth Scaffidi P, Gordon L, Misteli T (2005) The cell nucleus and aging: Tantalizing clues and hopeful promises. PLoS Biol 3(11): e395.

  47. My Long Journey To Antisense Oligos [ 1968 to 1993 ] And The Remaining Journey To Antisense Therapeutics Including Fountain of Youth [ 1993 to 2013 ? ] James Summerton, Ph.D. (Biochemistry) GENE TOOLS, LLC

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