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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 ? ]. James Summerton, Ph.D. (Biochemistry) G ENE T OOLS , LLC. Academic Phase. 1968 Joined Chris Mathews lab in Tucson - PowerPoint PPT Presentation
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My Long Journey To
Antisense Oligos[ 1968 to 1993 ]
And The Remaining Journey To
Antisense TherapeuticsIncluding
Fountain of Youth[ 1993 to 2013 ? ]
James Summerton, Ph.D. (Biochemistry)GENE TOOLS, LLC
Academic Phase
1968 Joined Chris Mathews lab in Tucson Conceived gene-blocking strategy for viruses1970
1972 Ph.D. in biochemistry: Chris Mathews1974 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, Corvallis1980 Switched to major-groove gene-blocking agents Left OSU, founded first gene-blocking company
Academic Phase
1968 Joined Chris Mathews lab in Tucson Conceived gene-blocking strategy for viruses1970
1972 Ph.D. in biochemistry: Chris Mathews1974 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, Corvallis1980 Switched to major-groove gene-blocking agents Left OSU, founded first gene-blocking company
First Scheme (1974-1978)
H
B
B
NN
O
NH2
HN
O
O
NN
O
N
N
NN
NN
O
H
H
H
H
HN
O O
Cl
NN
O
NHHN
NH2
O
O O
ClB
B
B
B
B
B
B
Antisense strand(carrier)
N
N
HNN
H2N
O
B
Sense strand(target)
Cross-linked carrier-target
Sequence-Specific Crosslinking Agents
Activated antisense strand
Academic Phase
1968 Joined Chris Mathews lab in Tucson Conceived gene-blocking strategy for viruses1970
1972 Ph.D. in biochemistry: Chris Mathews1974 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, Corvallis1980 Switched to major-groove gene-blocking agents Left OSU, founded first gene-blocking company
Second Scheme (1979-1982)
Major-Groove Binding Agents
BindingGCpair
BindingCGpair
BindingTApair
BindingATpair
AntiVirals, Inc.
Anti-Genes (1979-1982) AntiVirals, Inc.
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 agents1984 Dupont funding Devised Morpholino structural type1986 “Won” Oregon lottery1988
1990 Near-final Morpholino structural type
1992 Confirmed excellent properties1993 Discovered delivery problem
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 agents1984 Dupont funding Devised Morpholino structural type1986 “Won” Oregon lottery1988
1990 Near-final Morpholino structural type
1992 Confirmed excellent properties1993 Discovered delivery problem
AntiVirals, Inc.Third Scheme (1982-1993)
OHO B
OH
OH2N B
OH
OHN B
O
OHN B
O
O
Homopolymer binds
Heteropolymer does not bind
Heteropolymer binds
ON I
O
ON G
O
O
H
Competing structure
OO B
O
OO B
O
PO N
DNA analog
Cheap
Good activity
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 agents1984 Dupont funding Devised Morpholino structural type1986 “Won” Oregon lottery1988
1990 Near-final Morpholino structural type
1992 Confirmed excellent properties1993 Discovered delivery problem
Morpholinos From Ribonucleosides
Analogs derived from ribonucleosides
OHO B
OH OH
N
OO B
N
OO B
O
N
N
OO B
N
OO B
SO O
N
OO B
N
ON B
SO O
NH
OHO B
N
OO B
N
OO B
O
Binds DNAbut not RNA
Modeling suggested need for more flexible intersubunit link
Best
AntiVirals, Inc.
N
OO B
N
OO B
PO NR1
R2
Morpholino phosphorodiamidate structural types
=
B = A C G U
N
OO B
N
OO B
PO N
Near-final structure
Final structure
U T
T improves RNA binding affinityT allows sequencing by mass spectrometry
AntiVirals, Inc.
N
OO B
N
OO B
PO NR1
R2
NR1
R2
NH2
NH
N
N
N
O
NH
Morpholinos From Ribonucleosides
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 agents1984 Dupont funding Devised Morpholino structural type1986 “Won” Oregon lottery1988
1990 Near-final Morpholino structural type
1992 Confirmed excellent properties1993 Discovered delivery problem
MP S-DNA2’-O-Me
RNA PNAMorphsiRNA
efficacy
specificity
stability
in vivo delivery
cost of material
ease of assembly
splice modification
solubility
+ + + + + + + + + +
+ +
+ +
+ +
+ +
+ +
+ +
-
-
-
--
--
- - -
-
-
-----
+ ++ +
+ ++ +
+ +
+ ++ ++ +
+ +
+ + +++
+
+
++
+ -
+ +
O BO
O
O BO
O
PO
O BO
O
O BO
O
PO S
O BO
O
O BO
O
PO O
O
O
O BO
O
O BO
O
PO O
OH
OH N
O BO
N
O BO
PO N
NH
NO
B
O
N
NO
B
O
N
H
H
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
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
AntiVirals, Inc.
The Delivery ChallengeCell entry of oligomeric drugs
oligomerdrug
lateendosome
pH 5.5
lysosomepH 4.5
extracellularmedium pH 7.4
cytosolpH 7.5
degradedoligomer
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
AntiVirals, Inc.
watersoluble
watersoluble
lipidsoluble
EARLYENDOSOME
pH 7
LATEENDOSOME
pH 5
ENDOSOMALMEMBRANE
CYTOSOLpH 7
pH-Mediated Solubility Transition of Weak Acid Moieties
Molecular EngineFor Transporting Drugs Across Cell Membranes
James Summerton, Ph.D.ANTIVIRALS, Inc.
Delivery system 1: Molecular Engine AntiVirals, Inc.
Delivery system 1: Molecular Engine AntiVirals, Inc.
Octanol / Water Partitioning
Delivery system 1: Molecular Engine AntiVirals, Inc.
Molecular Engine: not enough power for Morpholinosso adapted for targeting acidic areas of tumors
Delivery system 1: Molecular Engine
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
AntiVirals, Inc.
Delivery system 2: Scrape Delivery
culture plate
adherent cell
scrapedcell
cargo molecules in medium
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
AntiVirals, Inc.
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
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
Morpholino
PEG-600
high-osmolarity sugar
release step
wash
Delivery system 3: Osmotic Delivery
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
Delivery system 4: Microinjection
Microinjection Into Eggs & Embryos
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
Morpholino
EPEI
Delivery system 5: Special Delivery
DNA
Delivery system
Rel
ativ
e m
agni
tude
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
Delivery system 6: Endo-Porter
Figure 1: Probable Endo-Porter mechanism
Delivery system 6: Endo-Porter
Delivery system 6: Endo-Porter
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
Delivery system 7: Vivo-Morphlinos
Delivery system 7: Vivo-Morphlinos
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
Delivery system 8: Receptor/Transport System (in progress)
Design objectives
cell levelhigh-affinity receptor on nearly all cellsefficient endocytosisefficient sortingefficient transport to cytosol
organ levelefficient transcytosis across blood-brain barrier
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
Fountain of YouthHutchinson-Gilford Progeria Syndrome
Nuclearmembrane:normal
Nuclearmembrane:HGPS
Fountain of Youth
Scaffidi P, Gordon L, Misteli T (2005) The cell nucleus and aging: Tantalizing clues and hopefulpromises. PLoS Biol 3(11): e395.
My Long Journey To
Antisense Oligos[ 1968 to 1993 ]
And The Remaining Journey To
Antisense TherapeuticsIncluding
Fountain of Youth[ 1993 to 2013 ? ]
James Summerton, Ph.D. (Biochemistry)GENE TOOLS, LLC