Download Nucleotide Modifications for Improved Messenger RNA Expression

Nucleotide Modifications for Improved Messenger RNA Expression
Anton P. McCaffrey1, Richard I Hogrefe1, Krist Azizian1, Alexandre Lebedev1, Dongwon Shin1, Julie R. Escamilla-Powers1, Brea Midthune1, Hiroko Yokoe2, Jonathan Kirschman3, Philip Santangelo3 and Joel Jessee2
1
TriLink BioTechnologies, San Diego CA 92121, USA, 2Molecular Transfer, Gaithersburg MD 20877, USA. 3Georgia Institute of Technology and Emory University Atlanta, GA 30332, USA.
EGFP
Limitations
• Innate immune response to unmodified mRNA
Solutions
• Chemical modification of mRNA can prevent innate immune
stimulation
• Pseudouridine or 5-methylcytidine/pseudouridine are current
industry standard
Luciferase Activity
10
30
20
1.0×10 6
10
0
oU
T
W
5m
o
N
5m
RN
A
oU
0
5m
T
A
eC
W
RN
o
40
2.0×10 6
0
N
50
3.0×10 6
PKR +/+ MEFs
Light Units
100000
PKR -/- ME Fs
10000
1000
100
10
1
Mock
AdMSC
HDF
HeLa
HuVec
NSC
SY5Y
35,000,000
Relative Light Unites
30,000,000
5moU
5moC/5moU
EGFP % Cells Transfected
100
25,000,000
20,000,000
PKR -/- ME Fs
10
15,000,000
1
PKR +/+ MEFs
PKR -/- ME Fs
1000000
100000
5moU
10,000,000
EGFP Mean Fluorescence
10000000
PKR +/+ MEFs
Fluorescence Units
Some Modifications Had No Activity
- 5fC
- 5hoC
- N6-Methyl-ATP
- N6-Me-2-amino-ATP
- N4-Methyl-CTP
1000000
% Cell Transfected
Cell Culture Evaluation of Modified mRNA
5moC/5moU
5moU
5moC/5moU
5,000,000
me1Ψ
5moU
Ψ
5hmC
5camU
5meC
5moC
wild type
30000
20000
10000
0
AdMSC
HDF
HeLa
HuVEC
NSC
SY5Y
5 Ψ
5m me
eC C
/Ψ
5 Ψ
5m me
eC C
/Ψ
5 Ψ
5m me
eC C
/Ψ
5 Ψ
5m m
e
eC C
/Ψ
5 Ψ
5m me
eC C
/Ψ
40000
5 Ψ
5m me
eC C
/Ψ
0
50000
Figure 5: Combinations of
Modifications are not Superior to Any
Single Modification for EGFP mRNA
Sequence Context
Conclusion
By comparing Figures 4 and 5 with
Figures 6 and 7 it is evident that
sequence context influences which
modifications are optimal.
35000
AdMSC
HDF
HeLa
HuVec
NSC
SY5Y
wild type
25000
20000
15000
5meC
5-hydroxycytidine
(5hoC)
Fluorescence
30000
5-formylcytidine
(5fC)
10000
5-hydroxymethylcytidine 5-methoxycytidine 5-methylcytidine N4-methylcytidine thienoguanosine 5-carboxymethylesteruridine
(5hmC)
(5moC)
(5meC)
(me4C)
(thG)
(5camU)
Stress granules are dense aggregates of RNP complexes that are
indicative of stalled translation.
control
45000
40000
N6-methyl-2-aminoadenosine N6-methyladenosine 5-carboxycytidine
(me6DAP)
(me6A)
(5caC)
Figure 8a: Modification of mRNA
Can Attenuate Stress Granuales
DAPI
Figure 1: Modified Bases Assessed
5000
5-methoxyuridine
(5moU)
N1-methylpseudouridine 5-methyluridine
(me1Ψ)
(5meU)
pseudouridine
(Ψ)
5 Ψ
5m me
eC C
/Ψ
5 Ψ
5m me
eC C
/Ψ
5 Ψ
5m me
eC C
/Ψ
5 Ψ
5m m
e
eC C
/Ψ
5 Ψ
5m me
eC C
/Ψ
5 Ψ
5m me
eC C
/Ψ
Ψ
0
5-hydroxymethyluridine
(5hmU)
20
% Stress Granule
4.0×10 6
• Several modified nucleotides enhance translation in a rabbit
reticulocyte lysate in vitro translation system
• In the six cell types tested, several modifications had
comparable or superior activity relative to pseudouridine
• Primary sequence influences activity of some modified mRNAs
• In most cases, adding a second modified nucleotide did not
increase activity over single modification
To identify novel patterns of chemical modifications for optimal
mRNA based therapeutics.
• Reduce innate immune responses and toxicity
• Maximize extent and duration of expression
5-formyluridine
(5fU)
30
Figure 7: Combination of
PseudoU/5meC Can Be Superior
to PseudoU Alone in the FLuc
Sequence Context
Objectives
diaminopurine
(DAP)
40
Luciferase
- Product of Molecular Transfer Inc.
- Cationic lipophilic nanoparticle
- Transfects a variety of cell lines with as little as 2 pg/cell of mRNA
Fluorescence
Applications
• Genome editing (Transposons, Cre, ZFNs, TALENs and CRISPR/
Cas9)
• Gene replacement
• Vaccines
50
Figure 9: Activity of Modified EGFP
and Luciferase mRNAs in PKR
Knockout MEFs
Figure 3: mRNA-In™ Transfection
Reagent
Cell Lines Tested
- Adipose mesenchymal cells (AdMSC)
- Adult human dermal fibroblast cells (HDF)
- Human cervical carcinoma cells (HeLa)/
- Human umbilical vein endothelial cells (HuVEC)
- Human neural stem cells (NSC)
- Human neuroblastoma cells (SY5Y)
Activity
% Stress Granule
1.1×10 6
1.0×10 6
9.0×10 5
8.0×10 5
7.0×10 5
6.0×10 5
5.0×10 55
4.0×10
3.0×10 5
2.0×10 5
1.0×10 5
0
5m
Fluorescence Intensity
RLU (Ffluc:Rluc)
0
Figure 4: EGFP Fluorescence with
Singly Modified mRNAs
• Does not have a risk of insertional mutagenesis
• Can transfect difficult cells such as non-dividing cells
• Is transient
Luciferase
Activity
10
Background
mRNA is a popular new tool for gene expression because it:
Figure 8b: Primary Sequence
Influences Stress Granule Formation
eC
20
Figure 6: Single Modifications in
Luciferase Have Different Activities
Than in the EGFP Sequence Context
% Stress Granule
Previously, a significant barrier to mRNA-based gene therapy had
been mRNA induced innate immune responses in transfected
cells. However, Kariko et al. showed that substitution of mRNAs
with pseudouridine and 5-methylcytidine dramatically reduces
innate mRNA immune recognition and highlighted the potential
for modified mRNA in the clinic. Activity and immunogenicity
of mRNA likely depends on the chemical modification pattern,
route of delivery, cell type/tissue transfected and possibly RNA
structure. Here we investigate a series of base modifications in
a variety of combinations in both EGFP and Firefly Luciferase
mRNA. After assessing incorporation of each nucleotide by T7 RNA
polymerase, we tested the translation potential of each modified
mRNA in rabbit reticulocyte lysates. The activity was further
evaluated in primary and immortalized cell lines transfected
with mRNA-In™. Though we observed some general trends, we
noted several instances where cell type influenced expression
levels. To determine the degree of innate immune stimulation,
we then assessed stress granule formation. Interestingly, for
some modifications, the primary sequence was an important
determinant of activity and stress granule formation. Finally, to
determine if protein kinase R (PKR) is important for the innate
response to modified mRNAs, expression levels were measured
in PKR deficient mouse embryonic fibroblasts. This investigation
expands our knowledge of the optimal chemical modifications for
maximum mRNA expression in different cell types
Figure 2: In Vitro Translation of
Modified mRNA in Rabbit Reticulocytes
% Stress Granule
In the last several years, in vitro transcribed mRNA has gained
traction as a potential new therapeutic agent to deliver genetic
information. mRNA provides several inherent benefits over
traditional plasmid- and virus-based methods for gene therapy.
One benefit is that mRNA is expressed in the cytoplasm and need
only cross one membrane. This facilitates higher transfection rates
and thus may be particularly useful for improving gene expression
in difficult to transfect, non-dividing cells. Additionally, in contrast to
plasmid or viral vectors, there is no risk of insertional mutagenesis
or subsequent oncogenesis upon mRNA transfection. Finally, the
transient nature of mRNA is desirable for a number of applications,
including cellular reprogramming, genome editing (ZFNs, TALENs
and CRISPR/Cas9) and vaccines.
5h
m m Ψ
C e1
/5 Ψ
5m hm
eC U
/
5m Ψ
5m
o
oC 5m U
5h / e
m 5m C
C e
/m U
w e1
ild Ψ
ty
5m
p
eC 5hm e
/m C
e
5m 1Ψ
5c oC
5m
am
eC
U
5m /5 5
c fC
5h oC am
m /m U
C e1
/5 Ψ
m
o
5c U
a
5h C
oC
th
ne
G
m
ga
6A
tiv
e
5
5mcon fU
t
5m eC rol
eC / thG
/5
fU
Abstract
GFP
G3BP
Merge
• Pseudouridine modified RNAs produced the fewest stress
granules
• Modifications that do not cause stress granules may still
activate PKR
Future Directions
• Test remaining modified RNAs in PKR -/- MEFs
• Test modifications in a Cas9 sequence context
• Assess whether HPLC purification influences performance
• Perform toxicity, dose response and cytokine measurements
• Conduct timecourse of expression to examine mRNA stability
• Assess structure/activity relationship studies to improve NTP
design
• Conduct single molecule mRNA/protein interaction studies to
determine interaction with innate immune sensors TLR7, MDA5,
RIGI
Contact
Dr. Anton McCaffrey, [email protected]
The Modified Nucleic Acid Experts
www.trilinkbiotech.com
™