Taming the Helix: The first 40 Years of RNA Therapeutics by Tod Woolf

Taming the Helix:
The First 40 Years of RNA Therapeutics
Tod Woolf, Ph.D.
Executive Director, Technology Ventures Office, BIDMC, (2019-Present)
Co-Founder and CEO, ETAGEN Pharma (2014-Present) (shareholder)
Business Advisor, Advirna (2010-Present) (shareholder)
Co-Founder, CEO, SAB, Ribozyme Pharmaceuticals (2006-2009)
Founder and CEO, Sequitur, (1996-2004)
Senior Scientist, RXi Pharmaceuticals (1993-1994)
Scientist, Genta (1992-1993)
Ph.D. Candidate, Harvard University (1987-1991)
Copyright 2020, Tod Woolf and the other cited copyright holders
Not for Distribution Outside of Recipient Organization

A Gene is the Instructions to Make a Protein:
DNA to RNA to Protein
Copyright IPIFINI, 2005
DNA: Double Helix
RNA: Single-stranded
temporary copy of DNA

RNA Therapeutics
Silencing mRNA
Therapeutic Editing™
Chemically-modified mRNA Therapeutics™ and Vaccines

RNA Therapeutics
Silencing mRNA
• Antisense Cleavers
• RNAi

5
Chemistry Toolbox used to Impart Nucleic Acids with Desirable Therapeutic Properties
Copyright, 2019
Phosphorothioates
• Chemical toxicity, Lower affinity
• Substrate for RNase H
• “Sticky”, enhances biodistribution and cell uptake
• High but not absolute nuclease stability(1-3 days)
2’ Modified Sugars (2’-O-mt, 2’F, LNAs... etc)
• High affinity
• Varied nuclease stability
• Low immune stimulation
Modified Nucleobases (pseudo U, G-Clamp… etc.)
• Can lower immune stimulation
• Can increase affinity
Neutral Modified Backbones (Phosphoramidates, PNAs, Morpholinos)
• Not recognized by cell machinery- No RNase H cleavage
• Extremely nuclease stable
• No immune stimulation
Unmodified DNA
• Very low endo and exonuclease stability
O P
O
S-
O
=
Linker/Conjugates
• Delivery ligands
• Exonuclease blockers

Paul Zamecnik Publishes First Antisense
Experiment in Mammalian Cells in 1978
• Paul Charles Zamecnik (1912-2009)
• Co-discovered transfer RNA
• First-ever Lasker Lifetime Achievement Award in 1996.
End-Blocked DNA
Low stability in cells
Stephenson ML, Zamecnik PC. Inhibition of Rous sarcoma viral RNA translation by a specific
oligodeoxyribonucleotide. Proc Natl Acad Sci U S A. Vol. 75 1978
KEY:
UNMODIFIED DNA
Conjugate

Antisense RNase H Cleaving Mechanism:
Oligo-Directed Destruction of Target RNA in Cells
RNase H
Target RNA
Antisense DNA Oligo

First Demonstrations of Antisense
and RNAi Off-Target Effects
Woolf, T., Melton, D., and Jennings, C. Specificity of
antisense oligonucleotides in vivo. Proceedings of the
National Academy of Science 89:7305-7309, 1992.
Birmingham, A. et al. .. KHVOROVA, A 3' UTR seed matches,
but not overall identity, are associated with RNAi off-targets.
Nat Methods 3, 199-204 (2006).
Charles
Jennings
Anastasia
Figure 1: Microarray analysis
identifies off-targeted genes.

Nuclease Resistant in humans (1-3 days)
Uptake into organs and cells
First Generation Phosphorothioate
DNA Antisense Therapeutics
Stein, C.A., Subasinghe, C., Shinozuka, K. and Cohen, J. "Physicochemical properties of phosphorothioate
oligodeoxynucleotides", Nucl. Acids Res., 1988, (16) 3209-3221.
• Uniform Phosphorothioate DNA Pursed by Ionis (Isis), Lynx, Hybridon and Genta
• ~1 gram per week systemic dose in humans
• One drug approved, all other major first generation trials failed due to lack of efficacy-toxicity
• Phosphorothioate linkages used with other modifications in most siRNA and antisense
Jack Cohen
Cy Stein
KEY:
PHOSPHOROTHIOATE DNA

RNase H-Mediated Target mRNA
Cleavage in Frog Oocytes with Gapmer
Dagle, Walder, Melton, Weeks and Woolf, 1991
Joe Walder
KEY:
UNMODIFIED DNA
NEUTRAL

Optimizing the Cell Efficacy of Synthetic Ribozymes . Thale C. Jarvis, Francine E. Wincott, Laverna J. Alby, James A. McSwiggen, Leonid
Beigelman, John Gustofson, Anthony DiRenzo, Kurt Levy, Melissa Arthur, Jasenka Matulic-Adamic, Alexander Karpeisky, Carolyn Gonzalez,
Tod M. Woolf, Nassim Usman, Dan T. Stinchcomb. Journal of Biological Chemistry 11/1996; 271(46):29107-29112.
WOOLF, TM, et al. "MODIFICATIONS REQUIRED FOR RNA OLIGONUCLEOTIDES TO EFFECTIVELY BIND MESSENGER-RNA TARGET SEQUENCES IN
CELLS." JOURNAL OF CELLULAR BIOCHEMISTRY. DIV JOHN WILEY & SONS INC 605 THIRD AVE, NEW YORK, NY 10158-0012: WILEY-LISS, 1995.
Low Potency
Hi Potency
Low Potency
Hi Potency
Unmodified RNA
KEY:
PHOSPHOROTHIOATE
UNMODIFIED RNA or DNA
2’ –O-Mt MODIFIED
Nuclease
In cell culture with Lipofectin
Defining the Rules for Stabilizing RNA Oligos in Cells

Sequitur Gapmers Used in Validating the Beta-Secretase
Tod Woolf, Amy Arrow, Roderick Dale. Three Component Chimeric Antisense Oligonucleotides1996 US Patent 5849902 A

48
Combinations of Chemistry Improve Antisense Potency 1000-Fold in Patients
Antisense Compound ID50 in vivo (mg/week)
Phosphorothioate (S-DNA):
GEN1
1500
S-DNA+2’ MOE wings:
GEN2
<150
S-DNA+ Replace MOE with
cET:
GEN2.5
<15
S-DNA +cET + GalNac:
GEN2.5LICA
<1.5
Stan Crooke
KEY:
PHOSPHOROTHIOATE
UNMODIFIED
2’ MOE or LNA MODIFIED
NEUTRAL
Nucleobase Modifications
Conjugate
Licenses &
Other
Technologies

RNA Therapeutics
Silencing mRNA
• Antisense Cleavers
• RNAi
mRNA Therapeutics and Therapeutic Editing are Trademarks of Tod Woolf, All Rights Reserved

RNAi Compounds
 Extraordinarily High Activity
 Specificity?
 No phosphorothioates required?
 Nuclease Stability?
 Uptake in vivo?????
Fire and Mello, Nature, Volume 391, 1998
Craig
Mello
Andrew
Fire
ALL
UNMODIFIED
siRNA
CLINICAL
TRIALS FAILED

0.0
0.2
TARGET
/
GAPDH
RNA
STEALTH RNAi TM
Standard siRNA
serum stability
0 4 24
8 30 48
STEALTH RNAi TM
Standard siRNA
hours
50% calf serum, 37oC
STEALTH RNAi TM
Standard siRNA
0 4 24
8 30 48
+ CTRL + CTRL
Phase Fluorescence
cytoplasmic stability
24 hours post-transfection
IRF-1
IKb
Viability
STEALTH RNAI
siRNA
Normalized
Value
Toxicity Markers
0
0.2
0.4
0.6
0.8
1
Interferon
PKR
IFIT1
less toxicity
FREEDOM TO OPERATE
Sequitur STEALTH RNAi
TM
>4,000 Literature Citations Using STEALTH RNAi
Woolf and Wiederholt, US Patent 9,592,250 2017, Filed 2003
Peggy Taylor
Kristin
Wiederholt
KEY:
UNMODIFIED RNA
2’ MODIFIED

sd-RNA: In Vitro and In Vivo Performance
Hepatocytes
primary mouse
ARPE-19
retinal pigment
epithelium
Macrophages
primary mouse
Keratinocytes
human primary
SH-SY5Y
neuroblastoma
Skin Eye Liver
Spinal
column
Delivery and silencing demonstrated in many different cell types
Human, Primate, Rat, Mouse, Adherent, Non-adherent, Primary,
Transformed
Efficient delivery of sd-RNA to multiple tissues in vivo upon
local and systemic administration
Lung
macrophage
s
KEY:
PHOSPHOROTHIOATE
UNMODIFIED RNA
2’ MODIFIED
Conjugate
Anastasia
Khvorova
Dmitry
Samarski

In Vivo Dose Dependent Silencing
Day 1 2 3
Biopsy
harvest
Incision
sd-RNA
injection
0
20
40
60
80
100
120
140
160
180
200
Target
Expression
%
of
PBS
↓78%
p = 0.003
↓72%
p = 0.005
10 30 100 300
Dermal Target (ug)
10 30 100 300
Non-Targeting Control (ug)
PBS
• 3 mm skin biopsies harvested and processed
• Data analyzed by QPCR and normalized to b-Actin
• Dosing in 200 ul volume
Pam Pavco
First Oligo Delivery Conjugate to Enter Clinical Trials
KEY:
PHOSPHOROTHIOATE
UNMODIFIED RNA
2’ MODIFIED
Conjugate

GENETIC MEDICINES
Revusiran Phase 2 Preliminary Study Results
19
Open label, multi-dose study in TTR cardiac amyloidosis patients
• Generally well tolerated
◦ Transient mild ISRs in 23% patients; 1 SAE (LFT increase to ~4x ULN in 1 patient, resolved
with continued dosing)
◦ No study discontinuations and no changes in renal function, other lab chemistries, or
hematologic parameters
Mean
(SEM)
%
Serum
TTR
Knockdown
(
Rel.
to
Baseline)
40 50 60
Days since first visit
Revusiran Dose Group
5.0 mg/kg 7.5 mg/kg
100
80
60
40
20
0
0 10 20 30
Revusiran (mg/kg), qd x5; qw x5
70 80 90 100
N=22
N=19
N=18
N=15
N=3
Treatment
Results as of October 3, 2014; Maurer, AHA, November 2014
N Individual Max
KD (%)
Mean ± SD Max
KD (%)
All 22 98.2 87.2 ± 9.1
5.0 mg/kg 19 97.7 86.4 ± 9.4
7.5 mg/kg 3 98.2 92.1 ± 5.4
Muthiah (Mano)
Manoharan
KEY:
PHOSPHOROTHIOATE
2’ MODIFIED
Conjugate
Max Planck
Licenses & Acquisitions

Self-Delivering RNAi Conjugates Companies
20

22
A Generalized Model for Therapeutic Editing™
RECOGNIZE
MOD
RECOGNIZE
MOD
mRNA Sequence Modification Activities:
1) Nucleobase Mod (e.g. Deamination)*
2) Exon Skipping (Kole)
3) Tran-splicing (Sullenger)
DNA Sequence Modification Activities
2) Cleavage and Ligation*
3) Recombination (Kmiec)
4) Mismatch Repair (Steer)
mRNA Target
DNA Target
*Tod Woolf Therapeutic Repair of Mutated Nucleic Acid Sequences. Nature Biotech (Review) 16: 341-4 1998

23
RECOGNIZE
MOD
RECOGNIZE
MOD
mRNA Target
DNA Target

24
1995 Publication on Therapeutic mRNA Editing™

Therapeutic mRNA Editing Demonstration with the
Endogenous Double-Stranded DNA Adenosine Deaminase
Woolf, et al. Toward the therapeutic editing of
mutated RNA sequences. PNAS 92, 1995
…GCAACUGCAGAUGCAUGGCGUAGCACUCGGC….
…GCAACUGCAGAUGCAU I GCGUAGCACUCGGC….
…GCAACUGCAGAUGCAUAGCGUAGCACUCGGC…. mRNA
Guide RNA Delivered to Cell
Mutant
Stop
Codon
Translation
Edited
Codon
From Stop
to TRP
Endogenous dsRNA
Adenosine Deaminase
Deamination of A
To Inosine (read as G)
GUCUACGUAUCGCAUCGUGA
GUCUACGUAUCGCAUCGUGA

First Demonstration of Therapeutic RNA
Editing in a Cell Model
Correction of a Dystrophin Stop Codon
Woolf, Chase and Stinchcomb Toward the therapeutic
editing of mutated RNA sequences. PNAS 92, 1995
Jennifer
Chase

mRNA Editing is Oligo Length and Chemistry Dependent
Toward the therapeutic editing of mutated RNA sequences. Woolf, et al. PNAS 92, 1995
Editing in rabbit ret. lysates
2’-O-mt
Phosphorothioate
2’-O-mt
Phosphorothioate

MF Montiel-Gonzalez et al. Correction of mutations within the cystic fibrosis transmembrane conductance
regulator by site-directed RNA editing, PNAS 2013
First Demonstration of Therapeutic RNA
Editing in a Human Cell Culture
Rosenthal Maria
Montiel-
Gonzalez

Companies are Currently Pursuing Therapeutic mRNA Editing by
Nucleobase Modification (BASE Editing)
29
ShapeTX

30
RECOGNIZE
MOD
RECOGNIZE
MOD
mRNA Target
DNA Target

Neutral Blockers:
Morpholinos
 High Activity
 Extraordinary Specificity
> 7 Days Stability
Dr. Jim Summerton
Eugene Stirchak, James Summerton, Dwight Weller, Uncharged stereoregular nucleic acid analogs: 2.
Morpholino nucleoade oligomers with carbamate internucleoside linkages, Nucleic Acids Research, 17, 1989
Antivirals, Inc.
(now Sarepta)
Antisense-Mediated
Mutated Exon
Skipping strategy
Ryszard Kole

Morpholinos: Extraordinarily Low Toxicity
WATER Morpho
200uM
THIOATE
2uM
One cell in two cell embryo microinjected
M. Whitman, Harvard Dental School and T. Woolf., 2000
KEY:
PHOSPHOROTHIOATE
NEUTRAL

P. Sazani, F. Gemignani, S, Kang, M. Maier, M. Manoharan, M. Persmark, D. Bortner & R. Kole. Systemically
delivered antisense oligomers upregulate gene expression in mouse tissues volume Nature
Biotechnology 20 (2002)
Exon Skipping Antisense Oligos Work In Multiple Mouse Tissues
Mano
Ryszard Kole
KEY:
PHOSPHOROTHIOATE
UNMODIFIED
2’ MODIFIED
NEUTRAL
Conjugate

Normal mdx/control mdx/PPMO
Three months, six intravenous injections of 30
mg/kg of the PPMO at biweekly intervals
Body-Wide Dystrophin Restoration in Mice with Systemic
Administration of 2nd Generation Self-Delivering Morpholinos Oligos
Bo Wu, Hong M. Moulton, Patrick L. Iversen, Jiangang Jiang, Juan Li, Jianbin Li, Christopher
F. Spurney, Arpana Sali, Alfredo D. Guerron, Kanneboyina Nagaraju, Timothy Doran, Peijuan Lu, Xiao , Qi Long
Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino
oligomerPNAS 2008, 105 (39) 14814-14819
Hong Moulton
Pat Iverson
KEY:
NEUTRAL
Conjugate

Systemically Administered Morpholino Shows
Activity in Human Muscles
.

36
Ionis 3rd Antisense Drug Spinraza Approved in
2016 for Spinal Muscular Atrophy
Administering oligomer therapeutics by injection
into CNS for severe indications
Frank Bennett
KEY:
PHOSPHOROTHIOATE
2’ MOE MODIFIED

37
RECOGNIZE
MOD
RECOGNIZE
MOD
mRNA Target
DNA Target

38
Endogenous Cellular DNA Repair Machinery
Genomic Nucleotide Repaired by Editing
Mutated Genomic Nucleotide
ETAMER™
Genomic DNA
Woolf, Nature Biotech 16: 341-4 1998
ETAMERs, can act by BER, primer incorporation or homologous
recombination mechanisms depending on chemistry
ETAMERs Repair Disease Causing Gene Defects
Bob Brown

39
Comparison of
ETAMER™
Versions
In Cell Culture
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Gen 2 Gen 2
Ctrl
Gen 1 Gen 1
Ctrl
Gen 2 Gen 2
Ctrl
Gen 3A Gen 3A
Ctrl
Gen 3B Gen 3B
Ctrl
Gen 3C Gen 3C
Ctrl
72mers
Editing
Efficiency
(%)
S-DNA +
2’ Mods
S-DNA +
MPs
S-DNA +
5mt-C
S-DNA
Ends
Unmod.
DNA
Unmodified
S-DNA Ends
S-DNA +2’ Mods
S-DNA+MPs
S-DNA+ 5mtC
KEY:
PHOSPHOROTHIOATES
UNMODIFIED DNA
2’ –O-mt MODIFIED
NEUTRAL Methyl Phosphonates
5mt-C Modifications
Tod Woolf, Alexander Lebedev and Richard Hogrefe. Compositions and Methods of
Treatment of Disease Using Editing Oligonucleotides. US Patent Application 2014

40
Days after cell culture
treatment
3rd Generation Editing Compounds can Direct Repair to
the Target DNA Strand
GEN3
Control Chemical Modification
GEN2
Correction
Efficiency
(%)
US Patent 7,258,854 B2, exclusively licensed to ETAGEN
One 5’ methyl C across from mismatched UG
Carmen Bertoni, Arjun Rustagi and Thomas A. Rando, Nucleic Acids Research, 2009, 37:7468–7482
KEY:
PHOSPHOROTHIOATE
UNMODIFIED DNA
Nucleobase Modifications
Conjugate

Editing Activity of ETAMERs Variants in Cell Culture
Rivera, Kmiec & Woolf, 2015
Copyright ETAGEN, 2015 Kmiec et al.US Patent 7,258,854 B2, exclusive to ETAGEN, and ETAGEN pending patents
Editing
Efficiency
Editing in Cell Culture
0
0.05
0.1
0.15
0.2
0.25
0.3
WT No
Phosphorothioates
MUT No
Phosphorothioates
WT 22/42
nucleotids
modified
MU 22/42
nucleotides
modified
GEN3 Variants
Co-Efficiency
KEY:
PHOSPHOROTHIOATE
UNMODIFIED DNA
2’ –O-mt MODIFIED
NEUTRAL
41

42
ETAMER Genome Editing Accumulates with Repeat Dosing
Relative
Correction
Efficiency
Reporter Gene Editing in Cell Culture
(Charlotte Andrieu-Soler, et al. NAR 2005 and Kmiec et al.US
Patent 7,258,854 B2, exclusive to ETAGEN)

Neutral Backbone Oligomers can form Triple-Stranded “Clamps”
“Chemical CRISPR”) that Slow Polymerases and enhance editing by
ETAMER
43
Primer
KEY:
NEUTRAL

44
ETAMERs Functionally Cure Beta-Thal in a
Mouse Model Without Programmable Nucleases
Four I.V. treatments over 8 days of encapsulated ETAMERs and Chemical CRISPR™
Days after last treatment
NEGATIVE CONTROL TARGETED
Hemoglobin
(g
dl–1)
Independent work with GEN2 ETAMERs
Bahal, R., Ali McNeer, N., Quijano, E. et al. In vivo correction of anaemia in β-thalassemic mice by
γPNA-mediated gene editing with nanoparticle delivery. Nat Commun 7, 13304 (2016).
US Patent 7,258,854 exclusively licensed to ETAGEN
KEY:
PHOSPHOROTHIOATE
UNMODIFIED DNA
NEUTRAL

45
3rd Generation ETAMER™ Chemical Modification
Improvements Based on Data External Data with ETAMERs
Internal Modifications:
• Protect against Endonucleases
• Reduce Immune Stimulation (TLR)
Editing Core
Modifications
Direct Editing to
Target Strand
Up to 100X
Exonuclease Protection
Up to 5X
5’ Chemistry
Up to
2X
3’ Chemistry
Consistent with
Editing
Modifications that Increase Affinity
Up to 2X
Optional Self-Delivery
ETAMER Conjugate
(i.e.. Cholesterol or Gal-Nac)

46
Advantages of Oligo-Only Editing
 Small size, facilitating delivery to tissues in vivo
 Protein-free design allows manufacture by chemical synthesis
 Non-immunogenic structure, allowing for multiple dosing
 No DNA cleavage, no insertions and deletions
 IP Protection and clear freedom to operate
Oligo-Only
ENGINEERED NUCLEASES
Competing Editing Approaches
CRISPR

.
Endogenous Cellular DNA Repair Machinery
Genomic DNA
dCAS9 crRNA with Partial DNA
Substitutions, acts as Guide and
Donor DNA
Proposed Use of dCas9 to Catalyze Strand Invasion of a
DNA Containing crRNA that Acts as a Guide and Donor
47

mRNA Transfection with Liposomes
Malone, Flegner and Verma, 1989 PNAS (not chemically modified)

mRNA as a Therapeutic
Potential Advantages Compared to DNA-based Gene Therapy or Protein Replacement:
• Gene-Free
• No integration or long term effects
• No immunogenic vector proteins
• mRNA is expressed more readily in primary cells (no nuclear delivery needed)
• Adjustable Kinetics (by modifying mRNA 3’ UTR)
• The protein is created in the cell with natural modifications, folding and processing
50 Sequitur Promotional Slide, 1998

Sequitur and Inex Enter into the First Alliance Focusing on
Developing MOD-mRNA for Therapeutics and Vaccines in 1998
• Sequitur was located in its founder’s basement in Natick, Massachusetts in 1998
• Inex (became Tekmira, now Arbutus) was a leading developer of liposome encapsulated DNA
for gene therapy
• Inex’s could not obtain expression of liposome encapsulated DNA in vivo
• Sequitur suggested to Inex that chemically modified mRNA may be more efficiently
expressed in vivo based on hypothetical arguments and data on antisense transfection
• The alliance resulted in data testing MOD mRNA and Unmodified mRNA in cells and animals
included in a Sequitur patent application published in 1999.
• The 2 year alliance was terminated at the end of the first year by Inex (Tekmira now Arbutus)
due to their financial position deteriorating

SEQUITUR: OmniVectTM
: First Marketed mRNA
Product by Sequitur for Gene Expression in Late
1990s
OmniVect Luciferase
Plasmid CMV-Luciferase
0
10000
20000
30000
40000
50000
60000
2 hr 4 hr
Hours Post Transfection
RLU
Zero Products Were Sold Due to Lack of Interest!
Allowed for uniform expression in difficult primary cells because
mRNA only needs to enter the cytoplasm and unlike DNA, mRNA
does not have to enter the nuclease and be transcribed

WHY STABLIZE RNA
• Serum nucleases
• Protect during cellular uptake process
• Increase duration of expression within cell
• More convenient to formulate and store
• [Reduces TLR activation]
Sequitur Promotional Slide, 1998

5
Chemistry Toolbox used to Impart Nucleic Acids with Desirable Therapeutic Properties
Copyright, 2019
Phosphorothioates
• Chemical toxicity, Lower affinity
• “Sticky”, enhances biodistribution and cell uptake
• High but not absolute nuclease stability(1-3 days)
2’ Modified Sugars (2’-O-mt, 2’F, LNAs... etc)
• High affinity
• Varied nuclease stability
• Low immune stimulation
Modified Nucleobases (pseudo U, G-Clamp… etc.)
• Can lower immune stimulation
• Can increase affinity
Unmodified RNA and DNA
• Very low endo and exonuclease stability
O P
O
S-
O
=

MOD-mRNA Experiments from 1998-1999
• Goal: Incorporation of 2’F C and U
• Rationale: C and U are the sites where RNases cleave
• Replacing these bases with 2’F derivatives will increase the
serum stability of mRNA
• Results have been reported with RNA oligos
• Question of translatability
• Progress
• Difficulty incorporating modified bases with Sp6 polymerase
• Luciferase transcript may be too long
• Incorporated into His-tag mini-RNA using T7 polymerase
Woolf, Wiederholt and Taylor, US Patent Appln. 20090093433, 1998

In vitro Transcription of Dimethyl G 5’ capped and 2’F Cytidine
in 1.2kb Luciferase mRNA
1 2
1) unmodified
2) 2’ F C Renilla LUC mRNA
Denaturing Agarose Gel, Ethidium Stain

Strategy for “mini-gene”
5’ UTR KozakAUG 6 His Tag Spacer X-press 3’UTR
-One version made by in vitro transcription (lacks Kozak sequence)
-One version completely synthetic
Sequitur 1998-1999

Detection of “mini-gene” peptide
• Development of ELISA for detection of mini-gene
• Plan to use 6 His tag and X-press epitope to
make a sandwich ELISA
• have obtained peptide to establish standard curve
• have synthetic RNA
Sequitur 1998-1999

2’F C and U
“mini-reporter” His Tag
1 2
1) unmodified
2) 2’ F C and U
Hogrefe, Woolf, Wiederholt and Taylor, 1999

MOD-mRNA Experiments
• Goal: Incorporation of 2’F C and U
• Rationale: C and U are the sites where RNases cleave
• Replacing these bases with 2’F derivatives will increase the
serum stability of mRNA
• Results have been reported with RNA oligos
• Question of translatability
• Progress
• Difficulty incorporating modified bases with Sp6 polymerase
• Luciferase transcript may be too long
• Incorporated into His-tag mini-RNA using T7 polymerase
2’F modified mRNA Failed to Translate, Approach Abandoned
Sequitur 1998-1999

MOD mRNA: Phosphorothioate Backbone
• Goal: Incorporate Alpha-thio nucleotides
• Rationale: Phosphorothioate DNA is resistant to nucleases.
Phosphorothioate RNA may be more stable than phosphodiester.
• Progress
• No incorporation using Sp6 polymerase
• Made T7 luc template
• Incorporated alpha-thio nucleotides
• QC by gel electrophoresis and in vitro translation
Sequitur 1998-1999

Incorporation of Alpha-Thio Nucleotides
into mRNA by In Vitro Transcriptions
1 2 3 4 5 6 7 10
8 9
1) a-S ATP
2) a-S ATP
3) a-S ATP
4) a-S ATP
5) all a-S
6) unmodified
7) 2:1 a-S:
unmodified
10) Molecular
markers

Dimethyl G 5’ Capped and
Individual Phosphorothioates in 1.2kb Renilla LUC
1 2 3 4 5
1) Thioate A
2) Thioate C
3) Thioate U
4) Thioate G
5) Unmodified
Renilla LUC mRNA

Mammalian Cell-Free Translation of
Phosphorothioate Containing mRNA
Thio A 1265/1236
Thio C 86095/93399
Thio G 99610/106602
Thio U 13287/11871
All Thio 58/64
No Thio 80188/88905
Background 73/65
LUC RLU’s, two replicas
Peggy Taylor
Kristin
Wiederholt

Adding Phosphorothioate and Unmodified Tail to
Capped LUC with Poly A Polymerase
1 2 3 4 5 6
1) Thioate Tail (30min Rxn.)
2) Thioate Tail (90min Rxn.)
3) Unmodified A Tail (30min Rxn)
4) Unmodified A Tail (60min Rxn)
5) Untreated
Renilla LUC mRNA

MODIFIED SENSE RNA ENHANCES EXPRESSION LEVELS
AND DURATION RELATIVE TO UNMODIFIED RNA
0
0.2
0.4
0.6
0.8
1
0 20 40 60 80
HOURS POST TRANSFECTION
A
C
B
Normalized
Renilla
Luciferase
Woolf, Wiederholt and Taylor, 1998

Modified mRNA Enhances Expression
Levels and Duration in Cell Culture
0
500
1000
1500
2000
2500
4 8 24
Hours Post Transfection
N=3
LUC
No Poly A Tail
60nt Poly A Tail
90nt Poly A Tail
No RNA
Peggy Taylor
Kristin
Wiederholt

Yield and Production of mRNA
• Third synthesis 3/19/98
• yield ~ 850 ug
• ~ 750 ug shipped to Tekmira (INEX) for mouse (IM) experiments

Intramuscularly Administered mRNA Expresses More
Protein than Plasmid DNA In Vivo
RNA
0
50
100
150
200
250
1 2 4 6 8 16 20 48
DNA PBS
Hours
PG LUC
PER GRAM
TISSUE
Woolf, Wiederholt and Taylor, 1998
• Expression of naked capped mRNA (with no chemical modifications)
injected into mouse muscle.
• Up to 20X more protein in cytoplasm with mRNA expression than
expression of DNA plasmid vector.
(formerly Inex)

In Vivo mRNA Delivery : Nanoparticles Yield High
Uniform mRNA Expression in Mouse Liver
70 Tekmira, 2015
• Red is expressed mCherry Protein
• Blue is nuclear stain
(formerly Inex)

3 of the Major COVID-19 Vaccine Programs use Sequence
and/or Chemically MOD mRNA
71

Modified mRNA Therapeutics™
Methods and Compositions Disclosed
in Sequitur 1997-98 Patent Application and Used in COVID Vaccines
Efficient 5’UTR
for Translation
Initiation
5’ Cap,
With optional
enhancements
Coding Region Optimized for
translation efficiency. Nuclease
sensitive sites removed
by changing 3rd nt in codons
3’ UTR for
long
message
half life in
cells (e.g.
beta
globin)
Poly A tail
for optimal
translation
and stability
Woolf, Wiederholt and Taylor, SENSE mRNA THERAPY US
Patent Appln. 20090093433, 1998, Sequitur, Inc
Deplete Uridines from the Sequence or
Incorporate Chemically Modified Uridines
In yet another embodiment, the
number of C's and/or U's in the
nucleotide sequence of an mRNA
molecule can be reduced by
incorporating analogs of C's and U's.
(deployed by Moderna and
Pfizer/BioNTech)
In another embodiment,
the number of C's and/or
U's is reduced by
substitution of one codon
encoding a particular
amino acid for another
codon encoding the same
or a related amino acid.
(used by CureVac)
the subject RNAs can be
made more nuclease
resistant by removing
nuclease sensitive
motifs…
wherein said mRNA
molecule encodes
an immunogen
which causes an
immune response in
a subject.
wherein the 5'
modification
comprises the
inclusion of a
modified
diguanosine
(m7)
…3' or 5' sequences which do
not normally flank said mRNA
molecule, an optimized Kozak
translation initiation
sequence, a coding region
depleted of C's or
U‘…(employed by Moderna)
..wherein said modification
comprises the inclusion of a
sequence affecting the secondary
structure of the mRNA. [e.g.
hairpins] (employed by Moderna)
delivery vehicles, e.g.,
cationic lipids,
uncharged lipids,
nanoparticles, or
liposomes…. can be
directly injected into
muscle.

What happened to Sequitur’s MOD-mRNA Program?
• The alliance with Inex was terminated at the end of the first year due to Inex’s deteriorating financial condition. Inex (Tekmira, Now
Arbutus) got back into mRNA therapeutics in subsequent years.
• Sequitur, which never raised outside investments, was funded by its antisense and siRNA functional genomics alliances and was sold to
Invitrogen (now ThermoFisher) for $10M in 2003 (which we thought was a lot of money at that time!)
• The Sequitur mRNA Therapeutics patent application was repeatedly rejected and the USPTO examiner repeatedly blocked the patent
application prosecution from entering into an appeal
• There was little or no licensing interest in Sequitur’s MOD mRNA Therapeutics and Vaccine Platform IP Over the next 11 years, until
Moderna inquired with Invitrogen about obtaining a license in 2010 (no license was consummated)
• Kariko and then Rossi demonstrated the improved properties of MOD mRNA with modified nucleobases, and identified pseudo U as an
excellent specific chemistry to deploy.
• Sequitur applications (acquired by Invitrogen, now ThermoFisher) were abandoned in the late 2010s)
• The breadth of the early Sequitur and the Vical applications led these applications to be cited as prior art repeatedly in the field, thus not
allowing any one company to dominate the mRNA Therapeutics and Vaccines IP landscape, and providing FTO for Moderna, BioNTech,
CureVac and others to pursue mRNA therapeutics and vaccines
• Moderna scientists used the expired Sequitur patent application as a guide for developing their MOD mRNA platform, and Modern and
BioNTech spent billions to build up and refine the mRNA Therapeutics and Vaccine Platform, heroically rapidly developing safe and effective
MOD mRNA vaccines for COVID-19
Lessons: it takes time, don’t give up on good ideas and sometime you can be too early

mRNA Therapeutics™ (Ribosome)
Successful Chemistries Direct Endogenous Cellular Machinery while
Imparting Nuclease Stability, Delivery and/or Reducing Toxicity
ETAMERs (DNA Repair)
Antisense (RNaseH or Spliceosome)
RNAi (RISC)
Therapeutic Editing™ of
mRNA (Deaminase)

LEADERS IN RNA THERAPEUTICS
Copyright IPIFINI, 2005
Cy Stein
Muthiah
(Mano)
Manoha
ran

Antisense Companies
Company Location Founded IPO Curr. Market Cap (March 2020) Platform Lead Product(s) (as of 10/20)
AVI/Sarepta Cambridge, MA 1985 1996 $9B
Morpholino Splice modulation
and gene therapy 2 Approved DMD
Gilead San Fran, CA 1987 1992 $100B
Lipophilic Self-Delivering
Conjugates
Left antisense biz early
Genta
Started in CA, finished in
NJ 1989 1991 Bankrupt
Methylphosphates and
Phosphorothioates Phase 3 failed, cancer
Sequitur Natick, MA 1996 -
Acquired by Invitrogen 2004,
now ThermoFisher Gapmers Research Products
Isis (Ionis) San Diego Area, CA 1989 1991 $7.5B Gampers and slice modulations 3 approved drugs
Hybridon (IDERA) Cambridge, MA 1990 1996 $470M
Immune Stimulation and
gapmers Antisense drugs failed
Lynx San Francisco, CA 1992 Private Defunct Phosphothiote Failed in clinical trials
Santaris Denmark 2003 Private Acquired by Roche 2014 $250M LNA gapmers and anti-miRNAs -
Prosensa The Netherlands 2002 2013
Acquired by Biomarin 2014
$680M Splice modulation Failed in clinical trials
Nogra Pharma Italy 2005 2014
Acquired by Celgene in 2014
$700M Phosphorothioates Failed in clinical trials
MiRagen Boulder, CO 2006 2016 Left Antisense Business 2020 Anti-miRNAs and miRNA mimics -
Mirna Austin, TX 2007 2015 Left Antisense Business 2020 Failed in clinical trials
Wave Life
Sciences Cambridge, MA 2012 2015 $300M Chiral phosphorothioates Phase 1
Stoke
Seattle, WA & Bedford,
MA 2018 Private -
Splice modulation
Pre-Clinical

siRNA Companies
Company Location Founded IPO
Curr. Market Cap
(March 2020)
Lead Product(s)
(as of 10/20)
Sequitur Acquired 1996 Acquired -
Stealth RNAi
marketed Research
Product
RPI/SIRNA IP Acquired by Alnylam 1996 2006
IP Rights acquired by
Alnylam for $175M
SIRNA Products
failed, licensed
technology used in
approved Alnylam
Products
Arrowhead Madison, WI and LA,CA 2000 1997 $3B 3 Phase 2
Alnylam Cambridge, MA 2004 2004 $12.2B 2 Approved
SILENCE London, England 1995 1995 $220M Phase 1
Rxi now Phio Westborough, MA 2008 2008 $13M
Phase 2
abandoned, Pre-
Clinical
MARINA Bothel, WA 2007 - Out of Business -
DICERNA Watertown, MA 2006 2014 $1.4B Phase 3
Atalanta Boston, MA 2019 - Private Pre-Clinical

mRNA Therapeutics™ and Vaccine Companies
Curr. Market Cap
(March 2020)
Lead Product(s)
(as of 10/20)
Sequitur Acquired 1996 Acquired - -
CureVac Tübingen, Germany 2000 Private $9.5B Phase 2
Arbutus (Tekmira, Inex) Vancouver, BC 1992 $190M
Moderna Cambridge, MA 2010? 2018 $28.B Phase 3
Translate BIO Cambridge, MA 2011 2018 $1.1b Phase 1/2
BioNTech Germany
$21.2 B(multiple
different
platforms) Phase 3

Genome Editing Companies
Company Location Founded
Curr. Market Cap
(March 2020) Platform Mechanism Products (Oct 2020)
Cellectis Paris and NYC 1999- IPO 2007 $515M TALENs Nuclease Cleave and mutate Phase 1
Sangamo Richmond, CA 1995- IPO 2000 $750M Zinc Finger Nucleae
Single Nucleotide
Sequence Change Phase 3
Precision Netherlands 2006 $380M CRISPR-Cas9 Cleave and Mutate Phase 1
Caribou/Intellia
San Fran and Cambridge
MA 2011 $680M CRISPR-Cas9 Cleave and Mutate Phase 1/2
Editas Cambridge, MA 2014 $1.2B CRISPR-Cas9 Cleave and Mutate Phase 1/2
CRISPR Ther.
Moved to Cambridge,
MA 2014 $2.7B CRISPR-Cas9 Cleave and Mutate Phase 1/2
LogicBio Cambridge, MA 2014 $170M AAV
Homologous
Recombination Phase 1/2
ETAGEN Cambridge, MA 2014 Private ETAMERs HR Pre-clinical
Homology Medicine Bedford, MA 2015 $815M AAV
Homologous
Recombination Phase 1/2
TruCode California 2019 Private ETAMERs and PNAs HR Pre-Clinical

Companies Pursuing Editing mRNA
Curr. Market Cap
(March 2020) Platform
Lead Product (Oct.
2020)
ProQR Leiden, Netherlands 2012 2014 $360M
Antisense Splice
Skipping- mRNA
editing
Phase 2/3 Antisense
Splice Skipping-
mRNA editing is Pre-
Clinical
ETAGEN Cambridge, MA 2014 Private - BASE Editing Pre-Clinical
Locana San Diego, CA 2016 Private -
RNA binding
Proteins Pre-Clinical
BEAM Cambridge, MA 2017 2020 $1.1B
Cas fused to
Deaminases Pre-Clinical
Korro Cambridge, MA 2019 Private -
Antisense RNA BASE
Editing Oligos Pre-Clinical
Shape Seattle, WA Private BASE Editing

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