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
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
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
First Scheme (1974-1978)
B
NH2
Sequence-Specific Crosslinking Agents
O
O
Cl
N
O
N
HN
O
O
B
Cl
O
NH
HN
B
H
NH2
N
N
O
Antisense strand
(carrier)
O
HN
Activated antisense strand
B
B
O
N
H
B
O
N
N
N
N B
H
O
N
N
O
B
H
N
N
N
HN
N
B
H
Cross-linked carrier-target
B
H2N
Sense strand
(target)
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
Second Scheme (1979-1982)
AntiVirals, Inc.
Major-Groove Binding Agents
Binding
GC
pair
Binding
CG
pair
Binding
TA
pair
Binding
AT
pair
Anti-Genes (1979-1982)
AntiVirals, Inc.
Commercial Phase
AntiVirals, Inc.
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
Commercial Phase
AntiVirals, Inc.
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
Third Scheme (1982-1993)
HO
O
B
H2N
O
B
AntiVirals, Inc.
HN
O
B
Homopolymer binds
OH
OH
O
O
HN
H N
Heteropolymer does
not bind
O B
I
O
O
O
Heteropolymer binds
O
N
O
O
G
Competing structure
O
B
O
DNA analog
O
Cheap
O P N
O
O
O
B
Good activity
Commercial Phase
AntiVirals, Inc.
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
Morpholinos From
Ribonucleosides
AntiVirals, Inc.
Analogs derived from ribonucleosides
HO
O
HO
B
O
O
O
B
B
Binds DNA
but not RNA
N
O
O
O
N
H
OH OH
B
N
Modeling suggested need for more flexible intersubunit link
O
O
O
B
N
N
O
O
B
O
O
B
O
N
B
B
N
N
N
O S O
O S O
O P N
O
O
O
O
O
N
B
N
O
B
O
R1
R2
O
N
N
Best
B
Morpholinos From
Ribonucleosides
AntiVirals, Inc.
Morpholino phosphorodiamidate structural types
O
O
R1
B
N
R2
N
O P N
O
Near-final structure
O
O
B
O
B
B=A
C
G
U
N
O
N
NH
Final structure
N
O P
N
R2
N
NH2
N
H
R1
O
=
U
T
N
O
N
B
T improves RNA binding affinity
T allows sequencing by mass spectrometry
Commercial Phase
AntiVirals, Inc.
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
MP
O
S-DNA
O
B
O
B
O
2’-O-Me
RNA
O
siRNA Morph
B
O
O
B
O
O
PNA
NH
B
O
B
N
O
O
O
O
P
O
O
B
P
O
O
B
O
P
O
O
O
O
B
O
P
OH
N
O
O
O
O
B
P
N
O
O
H
N
O
O
B
B
N
O
O
O
efficacy
O
S
O
O
O
O
H
N
N
OH
O
-
++
++
++
++
++
specificity
++
-
+
+
++
+
stability
++
-
+
-
++
++
solubility
-
++
++
++
++
-
cost of
material
-
-
-
+
++
++
ease of
assembly
+
++
+
-
++
++
splice
modification
+
-
++
-
++
++
in vivo delivery
-
-
-
-
-
-
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.
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
The Delivery Challenge
Cell entry of oligomeric drugs
oligomer
drug
extracellular
medium
pH 7.4
cytosol
pH 7.5
late
endosome
pH 5.5
lysosome
pH 4.5
degraded
oligomer
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
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
water
soluble
lipid
soluble
ENDOSOMAL
MEMBRANE
CYTOSOL
pH 7
water
soluble
Delivery system 1:
Molecular Engine
AntiVirals, Inc.
Delivery system 1:
Molecular Engine
AntiVirals, Inc.
Octanol / Water Partitioning
Delivery system 1:
Molecular Engine
Molecular Engine: not enough power for Morpholinos
so adapted for targeting acidic areas of tumors
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
Delivery system 2:
Scrape Delivery
AntiVirals, Inc.
cargo molecules in medium
scraped
cell
adherent
cell
culture plate
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
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
Delivery system 3: Osmotic Delivery
release step
wash
Morpholino
PEG-600
high-osmolarity sugar
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
Delivery system 5: Special Delivery
Morpholino
EPEI
Relative magnitude
DNA
Delivery system
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 level
high-affinity receptor on nearly all cells
efficient endocytosis
efficient sorting
efficient transport to cytosol
organ level
efficient 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 Youth
Hutchinson-Gilford Progeria Syndrome
Nuclear
membrane:
normal
Nuclear
membrane:
HGPS
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.
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