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 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