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My Long Journey To Antisense Oligos [ 1968 to 1993 ] And The Remaining Journey To Antisense Therapeutics Including Fountain of Youth [ 1993 to 2013 ?

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Presentation on theme: "My Long Journey To Antisense Oligos [ 1968 to 1993 ] And The Remaining Journey To Antisense Therapeutics Including Fountain of Youth [ 1993 to 2013 ?"— Presentation transcript:

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2 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

3 Academic Phase 1968 Joined Chris Mathews lab in Tucson Conceived gene-blocking strategy for viruses 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 Academic Phase 1968 Joined Chris Mathews lab in Tucson Conceived gene-blocking strategy for viruses 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

5 First Scheme ( ) H Antisense strand (carrier) Sense strand (target) Cross-linked carrier-target Sequence-Specific Crosslinking Agents Activated antisense strand

6 Academic Phase 1968 Joined Chris Mathews lab in Tucson Conceived gene-blocking strategy for viruses 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

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

8 Anti-Genes ( ) AntiVirals, Inc.

9 Commercial Phase Antisense Oligos ( ) 1980 Founded first gene-blocking company: ANTIVIRALS, Inc 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 Near-final Morpholino structural type 1992 Confirmed excellent properties 1993 Discovered delivery problem

10 AntiVirals, Inc. Commercial Phase Antisense Oligos ( ) 1980 Founded first gene-blocking company: ANTIVIRALS, Inc 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 Near-final Morpholino structural type 1992 Confirmed excellent properties 1993 Discovered delivery problem

11 AntiVirals, Inc. Third Scheme ( ) Homopolymer binds Heteropolymer does not bind Heteropolymer binds

12 Competing structure DNA analog Cheap Good activity

13 AntiVirals, Inc. Commercial Phase Antisense Oligos ( ) 1980 Founded first gene-blocking company: ANTIVIRALS, Inc 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 Near-final Morpholino structural type 1992 Confirmed excellent properties 1993 Discovered delivery problem

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

15 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 AntiVirals, Inc. Morpholinos From Ribonucleosides

16 AntiVirals, Inc. Commercial Phase Antisense Oligos ( ) 1980 Founded first gene-blocking company: ANTIVIRALS, Inc 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 Near-final Morpholino structural type 1992 Confirmed excellent properties 1993 Discovered delivery problem

17 MP S-DNA 2’-O-Me RNA PNAMorphsiRNA efficacy specificity stability in vivo delivery cost of material ease of assembly splice modification solubility

18 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 G ENE T OOLS, LLC AntiVirals, Inc.

19 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 G ENE T OOLS, LLC AntiVirals, Inc.

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

21 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 G ENE T OOLS, LLC AntiVirals, Inc.

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

23 Delivery system 1: Molecular Engine AntiVirals, Inc.

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

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

26 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 G ENE T OOLS, LLC AntiVirals, Inc.

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

28 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 G ENE T OOLS, LLC AntiVirals, Inc.

29 Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started G ENE T OOLS, 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) Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

30 Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started G ENE T OOLS, 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) Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

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

32 Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started G ENE T OOLS, 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) Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

33 Delivery system 4: Microinjection Microinjection Into Eggs & Embryos

34 Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started G ENE T OOLS, 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) Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

35 Morpholino EPEI Delivery system 5: Special Delivery DNA Delivery system Relative magnitude

36 Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started G ENE T OOLS, 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) Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

37 Delivery system 6: Endo-Porter Figure 1: Probable Endo-Porter mechanism

38 Delivery system 6: Endo-Porter

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40 Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started G ENE T OOLS, 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) Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

41 Delivery system 7: Vivo-Morphlinos

42 Delivery system 7: Vivo-Morphlinos

43 Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started G ENE T OOLS, 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) Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

44 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

45 Commercial Phase Antisense Therapeutics ( 1997 – 2013 ? ) 1997 Started G ENE T OOLS, 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) Delivery: Vivo-Morpholinos (Yongfu Li) (7) 2009 Delivery: Receptor/transport system devised (8) Delivery: Receptor/transport system completed (2013 ?) “Fountain of Youth” application pending delivery

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

47 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.

48 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


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