CleanCap® Co-transcriptional Capping Streamlines mRNA Manufacturing Dongwon Shin1, Krist T. Azizian2, Jordana M. Henderson1, Richard I. Hogrefe1, Alexandre Lebedev1, Michael Houston1, and Anton P. McCaffrey1

1TriLink BioTechnologies LLC, San Diego, CA 92121, USA 2Synthetic Genomics, Inc., La Jolla, CA 92037, USA

AbstractMessenger RNA (mRNA) therapy is a popular platform technology for expressing proteins in cells or in vivo because there is minimal risk of insertional mutagenesis. mRNA transfection is used to express proteins for genome editing, protein replacement, vaccines and antibody expression. To avoid an innate immune response, transfected mRNAs should mimic the 5’ cap structure of non-immunogenic endogenous mRNAs.

During eukaryotic RNA capping, Cap 0 (m7GpppN) is formed as an intermediate. Methylation of the 2’-O position of the first cap-proximal nucleotide forms Cap 1 (m7GpppN

mN). In ~50% of transcripts, the 2’-O

position of the second cap-proximal nucleotide is also methylated to form Cap 2 (m7GpppN

mN

m). N6-methylation of adenosine at the first

cap-proximal nucleotide (m7Gpppm6Am

N) is the second most frequently found modification in mRNA and occurs in conjunction with Cap 1 (and potentially Cap 2).

The immunogenic role of mRNA caps requires elucidation. Viral attenuation occurs after deleting methyltransferases that RNA viruses encode to convert Cap 0 to Cap 1. IFITs bind Cap 0 and activate antiviral translational repression. Thus, Cap 1 (and possibly Cap 2) marks endogenous mRNAs as “self” RNAs. The role of Cap 2 and Cap 1 (m6A) is poorly understood because such capped mRNAs have not been produced synthetically at scale. In a recent study, Cap 1 (m6A) caps may increase stability and translation while decreasing de-capping of mRNA (Mauer et al., Nature 2017, 541, 371-375).

Traditional co-transcriptional capping utilizes ARCA (Anti-Reverse Cap Analog) to produce immunogenic Cap 0 with poor capping (~70%) and low yield. Post-transcriptional enzymatic capping to produce Cap 0 or Cap 1 is hindered by highly structured 5’ ends, requires further purification and is expensive. Methods to produce Cap 2 mRNAs have not been commercially available. We developed CleanCap®, a novel co-transcriptional capping method to yield Cap 0, Cap 1, Cap 2, Cap 1 (m6A) or unnatural caps (Figure). Capping with CleanCap is reproducibly efficient (90-99%), less expensive than enzymatic capping and is done in a “one-pot” reaction without additional purification. In addition, CleanCap co-transcription method yields higher amount of capped mRNA than other methods including ARCA and enzymatic. Our studies in a THP-1 Dual monocyte cell line indicate that these various CleanCap mRNAs exhibit altered expression and immunogenicity. Further in vivo studies to characterize these mRNAs are ongoing.

Background: Why mRNA Therapeutics?

mRNA is a popular new tool for gene expression » Does not have a risk of insertional mutagenesis

» Can transfect difficult cells such as non-dividing cells

» Is transient

Applications » Genome editing (Transposons, Cre, ZFNs, TALENs and CRISPR/Cas9)

» Gene replacement

» Vaccines

Limitations » Innate immune response to unmodified mRNA

Solutions » Proper capping

» Chemical modification of mRNA can prevent innate immune stimulation

» Removal of dsRNA

Innate Immune Sensors Recognize mRNATransfection of cells with unmodified RNAs can lead to cell death due to activation of innate immune pathways

Toll-like receptors 3, 7, & 8 recognize different RNA forms » Found in endosomes where some viruses enter cells

Cytosolic sensors » Protein Kinase R (PKR): dsRNA

» MDA5: long dsRNA

» IFITs: unmethylated cap structures

» RIG-I: 5’-triphosphate

Figure 1: Cap 0, Cap 1 and Cap 2 Structures of 5’-Ends of mRNAs Eukaryotic mRNAs have a Cap 1 or Cap 2 structure.

Sensing of proper cap structure is thought to be involved in self/non-self RNA recognition.

Co-transcriptional capping with CleanCap (Cap 1) helps evade an immune response

Figure 3: mRNA Capping Assay: Enables Quantitation of Multiple Enzymatic Steps

Figure 5: CleanCap Analogs Expand the Range of 5’ Sequences that can be Used to Initiate T7 RNA Polymerase

Conclusions » CleanCap is a novel co-transcriptional capping method

» Very high and consistent capping efficiencies obtained with CleanCap

» CleanCap is an attractive, cost effective alternative to enzymatic or ARCA capping of mRNA

» CleanCap allows novel cap forms that were not previously accessible such as Cap 2 and Cap 1 (m6A)

» Cap 1 and Cap 1 (m6A) Cap RNAs are more active than Cap 0 RNAs in vivo

» Cap 1 (m6A) Cap alters activity in vivo and may extend persistence of m6Am

capped RNAs

» m6Am

G RNAs are de-capped more slowly than Am

G RNAs

» The identity of the second cap proximal nucleotide (m7Gpppm6Am

N) influences the rate of Dcp2 mediated de-capping

» m6Am

G displays the lowest de-capping rate for all capped forms

» Nudt12 and DXO selectively de-cap Cap 0 but not Cap 1 or Cap 2 RNAs

ContactAnton McCaffreyamccaffrey@trilinkbiotech.com

The Modified Nucleic Acid Experts®

www.trilinkbiotech.com

Figure 11: Nudt12 and DXO Selectively De-Cap Cap 0 but Not Cap 1 or Cap 2 RNAs

Figure 9: Protein Expression for Cap 0, Cap 1, Cap 2 or Cap 1 (m6A) HPLC Purified Luciferase mRNAs in Mice

Function of mRNA Cap StructuresmRNA cap structures are involved in modulating

» Nuclear export

» Splicing

» Turnover

» De-capping

Cap 1 and Cap 2 are important for self/non-self recognition by the innate immune system

» Cap 0 recognized as foreign

» IFITs recognize non-methylated caps

» Cap 1 methylation reduces binding to pattern recognition receptors

» Role of Cap 2 is largely unexplored because it was not possible to easily generate Cap 2 RNAs until now

Time (min)

Figure 7: Cap 1 Capping Assay

Uncapped

Cap 1

Time

Figure 8: Cap 1(m6A) Capping Assay

Figure 4: Anti-Reverse Cap Analog (ARCA) Capping is Inefficient

20.0

ARCA FLucARCA (Cap 0)

Transcription Stuttering

Uncapped

1.7E+008

1.4E+008

1.0E+008

6.9E+007

3.4E+007

0.0E+0006.53.0 10.0 13.50 17.0 20.0

Cap 0 CleanCap

Cap 1 CleanCap

Cap 1(m6A) CleanCap

Cap 2 CleanCap

Cap 0 is inferior to Cap 1, Cap 2, and Cap 1 (m6A) at 6 and 24 hours. Note: ARCA also yields Cap 0.m6A

m mRNAs are significantly superior to Cap 1 and Cap 2 mRNAs at 6 and 24 hours.

Uncapped

Cap 1 (m6A)

Time

Data courtesy of Samie Jaffrey (Cornell) and Mike Kiledjian (Rutgers)

Figure 10: In Vitro De-Capping with Dcp2 is Decreased with m6A Capped RNAs. Identity of First and Second Cap-Proximal Influences De-capping

Conclusions for Cap 1 m7Gpppm6Am

G is de-capped more slowly than Cap 1 m7GpppAm

G

» The identity of the second cap proximal nucleotide m7Gpppm6Am

N influences the rate of Dcp2 mediated de-capping

» m6AG displays the lowest de-capping rate for all capped forms

32P labeled capped oligonucleotides with different cap forms and 5’ sequences were de-capped in vitro with purified capping enzymes

Data courtesy of Samie Jaffrey (Cornell) and Mike Kiledjian (Rutgers)

m7 G

pppAG

A mG

m6 AA

m6 AC

m6 AG

m6 AU

m6 A m

Am

6 A mC

m6 A m

Gm

6 A mU

m6 A m

A mm

6 A mC m

m6 A m

G mm

6 A mU m

0.0

0.5

1.5

1.0

2.0

Rat

io o

f d

e-c

app

ing

Cap 1 Cap 2Cap 0

Cap 1 Cap 2Cap 0

0.0

0.5

1.5

1.0

m7 G

pppAG

m6 AA

m6 AC

m6 AG

m6 AU

m6 A m

A

m6 A m

C

m6 A m

G

m6 A m

Um

6 A mA m

m6 A m

C mm

6 A mG m

m6 A m

U m

Cap 1 Cap 2Cap 0

0.0

0.5

1.5

1.0

Figure 2: N6-methyladenosine Methylated Caps: Regulate Translation and mRNA Stability

Reversible methylation of m6Am

in the 5’ cap

controls mRNA stability

Mauer and Jaffrey et al.

Nature. 2017 541(7637):371-375

B1-3 = A, C, G, U or m6A (B1)

Cap 0: R1 and R2=H

Cap 1: R1=CH3; R2=H

Cap 2: R1=CH3; R2=CH

3

Figure 6: Comparison of Capping Methods

» Guanyl transfer reaction (Cap)

» N7 methylation of 5’-Guanosine (Cap 0)

» 2’-O-methylation (Cap 1)

» Phosphatase step

Enzymatic Cap 1 FLuc

Cap 1

Cap 0

Cap uncappedtriphosphate

uncappedmonophosphate

2.7E+007

2.2E+007

1.6E+007

1.1E+007

5.4E+006

0.0E+00016.015.0 17.0 18.0 19.0

Inte

nsi

ty

cap poly A

cap poly A

LC-MS

Cap

Cap

poly A

poly A

LC-MS

DXO

Nudt12

6 H o u r s

No

rma

liz

ed

to

To

tal

Pro

tein

Ca p

0

Ca p

1

Ca p

2

Ca p

1

m6 A

PB

S

0

2 , 0 0 0

4 , 0 0 0

6 , 0 0 0

8 , 0 0 0

1 0 , 0 0 0

2 4 H o u r s

No

rma

liz

ed

to

To

tal

Pro

tein

Ca p 0

Ca p 1

Ca p 2

Ca p 1

m

6 A

PB

S

0

1 0 0

2 0 0

3 0 0

a) At 6 and 24 hours Luciferase Protein was Measured in Liver by Western Blot

WT

5’-AGG

Not HPLC purified 5-MoU is showed similar activity to HPLC purified WT mRNAC

ap

1

Ca

p 1

m

6 A

Ca

p 1

Ca

p 1

m

6 A

PB

S

0

2 × 1 0 1 1

4 × 1 0 1 1

6 × 1 0 1 1

8 × 1 0 1 1

6 H r P o s t D o s e

To

tal

Flu

x (

p/

s)

* * *

* ** *

Ca

p 1

Ca

p 1

m

6 A

Ca

p 1

Ca

p 1

m

6 A

PB

S

0

2 × 1 0 1 1

4 × 1 0 1 1

6 × 1 0 1 1

8 × 1 0 1 1

2 4 H r P o s t D o s e

To

tal

Flu

x (

p/

s)

* * ** * *

* **

WT

5’-AGG

5-MoU, not HPLC

Dunnett’s multiple comparisons test * p<0.05 **p<0.01 ***p<0.001

Cap 0 is inferior to Cap 1 and Cap 2 at 6 and 24 hours.

Ca

p 0

Ca

p 1

Ca

p 2

Ca

p 0

Ca

p 1

Ca

p 2

r ef .

PB

S

0

1 × 1 0 1 1

2 × 1 0 1 1

3 × 1 0 1 1

4 × 1 0 1 1

5 × 1 0 1 1

6 h r P o s t - D o s e

To

tal

Flu

x (

p/

s)

* * * *

* *

* * * *

Ca

p 0

Ca

p 1

Ca

p 2

Ca

p 0

Ca

p 1

Ca

p 2

r ef .

PB

S

0

5 × 1 0 1 0

1 × 1 0 1 1

1 . 5 × 1 0 1 1

2 4 h r P o s t - D o s e

To

tal

Flu

x (

p/

s)

* * * *

* *

*

WT

5’-AUA

5-MoU

ref. Cap 1 WT m7GpppAm

GG Dunnett’s multiple comparisons test* p<0.05 **p<0.01 ***p<0.001

b) In Vivo Time-course of Luciferase Expression in Liver-Bioluminescence Imaging

c) In Vivo Time-Course of Luciferase Expression in Liver-Bioluminescence Imaging

WT

5’-AUA

5-MoU

To

tal F

lux

(p/s

)

5x1011

4x1011

3x1011

2x1011

1x1011

0

6hr Post-Dose 24hr Post-Dose

To

tal F

lux

(p/s

)

1.5x1011

5x1011

1x1011

0

Cap 0

Cap 1

Cap 2

Cap 0

Cap 1

Cap 2 ref.

PBS.

Cap 0

Cap 1

Cap 2

Cap 0

Cap 1

Cap 2 ref.

PBS.

To

tal F

lux

(p/s

)

6x1011

4x1011

2x1011

0

6hr Post-Dose 24hr Post-Dose

To

tal F

lux

(p/s

)

Cap 1

Cap 1 m

6 ACap 1

PBS

Cap 1 m

6 A

8x1011

Cap 1

Cap 1 m

6 ACap 1

PBS

Cap 1 m

6 A

6x1011

4x1011

2x1011

0

8x1011

WT

5’-AGG

5-MoU, not HPLC

Cap 1

Cap 2PBS

Cap 1 m

6 A

6 hours 24 hours

Cap 0

Cap 1

Cap 2PBS

Cap 1 m

6 A

Cap 0

10,000

8,000

6,000

4,000

2,000

0

300

200

100

0

No

rmal

ize

d t

o T

ota

l Po

tein

No

rmal

ize

d t

o T

ota

l Po

tein

WT

5’-AGG

CappingMethod

Capping Efficiency

Cost/mg Capped RNA

Cap 2 Achievable

m6AmAchievable

Purification Required Between

Transcription and Capping

CappingInhibited by

5’ End Structure

Enzymatic Variable High No No Yes Yes

ARCA ~70 % Moderate No No No No

CleanCap ~90-99% Low Yes Yes No No

CappingMethod

Capping Efficiency

Cost/mg Capped RNA

Cap 2 Achievable

m6AmAchievable

Purification Required Between

Transcription and Capping

CappingInhibited by

5’ End Structure

Enzymatic Variable High No No Yes Yes

ARCA ~70 % Moderate No No No No

CleanCap ~90-99% Low Yes Yes No No

Capping

Method

Capping

Efficiency

Cost/mg

Capped RNA

Cap 2

Achievable

Cap 1 (m6A)

Achievable

Capping

Inhibited by

5’ End

Structure

m7 G

pppAG

m6 AA

m6 AC

m6 AG

m6 AU

m6 A m

A

m6 A m

C

m6 A m

G

m6 A m

Um

6 A mA m

m6 A m

C mm

6 A mG m

m6 A m

U m