Chemical Design of Oligonucleotides That Support Targeted
RNA Editing in the CNS of Non-Human Primates
Chikdu Shivalila, Genliang Lu, Christopher Acker, Ian Harding, Jigar Desai, Jack Godfrey, Naoki Iwamoto, Pachamuthu Kandasamy, Hui Yu, Tomomi Kawamoto, Jake Metterville, Megan Cannon, Erin Purcell-
Estabrook, Alyse Faraone, Prashant Monian, Stephany Standley, Jesse Turner, Ryan Yordanoff, Fangjun Liu, Hailin Yang, Michael Byrne, and Chandra Vargeese
Wave Life Sciences, Cambridge, MA, USA
SUMMARY
RESULTS
Figure 3. Impact of AIMer optimization on editing
Figure 4. AIMers facilitate RNA editing in the CNS
Figure 5. ADAR editing for MECP2R168X protein restoration
• Leveraging PRISM™, our discovery and drug development platform, |
we developed relatively short oligonucleotides that elicit A-to-I RNA |
editing with high efficiency using endogenous ADAR enzymes, which |
we call AIMers (Figure 1A).1-3 |
• Using N-Acetylgalactosamine(GalNAc)-modified AIMers with a |
stereopure chimeric phosphodiester (PO)/ phosphorothioate (PS)/ |
phosphoryl guanidine (PN) backbone pattern, we previously |
demonstrated up to 50% editing in nonhuman primate (NHP) liver.2 |
• Here, we have further optimized AIMer design by identifying base, |
sugar, and backbone modification patterns that improve editing across |
target and nearest neighbor sequences. |
Figure 2. AIMer base, sugar and backbone modifications enhance editing efficiency across nearest neighbor combinations in cells
A | Approach to Improving Editing Efficiency | ||||
Orphan site | Edit region | ||||
AIMer | 3′ | 5′ | N-3-uridine | ||
NCN | |||||
HN | |||||
Target RNA | |||||
5′ | XAX | 3′ | O N O | ||
Nearest Neighbors | |||||
Sugar |
1 | Optimize orphan site base | 5′ | 3′ | Cytosine | N3U |
efficiency in vitro
A | Cell-free System | Ugp2 Editing | ||||||||
100 | Primary Mouse Hepatocytes | |||||||||
**** | ||||||||||
50 | **** | **** | ||||||||
**** | ||||||||||
40 | ||||||||||
80 | **** | |||||||||
**** | ||||||||||
Editing | 30 | **** | ||||||||
20 | **** | |||||||||
Editing | 60 | * | ||||||||
% | 10 | ns | ||||||||
0 | 6 | 24 48 72 96 | 6 | 24 48 72 96 | 6 | 24 48 72 96 | 6 | 24 48 72 96 | ||
% | Hours | |||||||||
40 | Stereorandom PS | Stereorandom PN | Stereopure PS | Stereopure PN | ||||||
AIMer Abundance |
A | In vitro Editing in CNS Cell Lines | ||||||||||
100 | ACTB | 100 | UGP2 | ||||||||
Editing | 80 | Editing | 80 | ||||||||
60 | 60 | ||||||||||
% ACTB | 40 | % UGP2 | 40 | ||||||||
20 | 20 | ||||||||||
0 | 0 | ||||||||||
0.01 | 0.1 | 1 | 10 | 100 | 0.01 | 0.1 | 1 | 10 | 100 | ||
Concentration (μM) | Concentration (μM) | ||||||||||
iNeurons | iAstrocytes |
- The Problem
Top 8 Hotspot Rett Syndrome (RTT) Mutations
10 | T158M R168X | Nonsense | ||||||||||
(%)Frequency | R106W | R133C | R255X R270X | R294X R306C | ||||||||
Missense | ||||||||||||
8 | ||||||||||||
6 | ||||||||||||
4 | ||||||||||||
2 | ||||||||||||
N-term | MBD | ID | TRD | C-term | ||||||||
1 | 78 | 162 | 207 | 310 | 487 |
Methyl-CpG Binding Protein 2 (MECP2)
The Approach
Amino Acid | Arg | Glu | Glu | |||||||
Normal | mRNA | C | G | G | C | G | A | G | A | G |
DNA | C | G | G | C | G | A | G | A | G | |
G | C | C | G | C | T C | T | C | |||
MECP2 | ||||||||||
RTT | Amino Acid | Arg | STOP | |||||||
mRNA | C | G | G | U | G | A | G | A | G | |
(nonsense) | ||||||||||
DNA | C | G | G | T | G | A | G | A | G | |
G | C | C | A | C | T C | T | C | |||
AIMer | MECP2R168X | |||||||||
RNA- | Amino Acid | Arg | Trp | Glu | ||||||
mRNA | C | G | G | U | G | I | G | A | G | |
edited | ||||||||||
C | G | G | T | G | A | G | A | G | ||
DNA | ||||||||||
G | C | C | A | C | T C | T | C | |||
MECP2R168W |
MECP2 Coregulatory
Protein Binding
293T Cells, Transfected
MECP2 | |
Mock | WT R168W |
90 | FLAG |
70 | |
MECP2 |
NCoR1
260
90
70TBLR1
IgG Heavy chain
50HDAC3
• Here, we show that AIMer RNA base editing technology is applicable in |
the central nervous system (CNS). AIMers support editing of |
housekeeping RNA in neurons and astrocytes in vitro, and |
unconjugated AIMers broadly direct durable RNA editing across the |
CNS of mice and NHPs. |
• AIMers with optimized chemistry support editing of a disease-relevant |
transcript in neuronal cells. MECP2 AIMers direct RNA editing to |
2 | Optimize sugar and backbone | AIMer-S | AIMer-D | ||
modification pattern outside 5′ | 3′ | ||||
Pattern | Pattern | ||||
the edit region | |||||
B | Orphan Site Base | C | Sugar and Backbone |
5′ | AIMer | 3′ | 5′ | 3′ |
20 | Primary Mouse Hepatocytes | ||||||
6 hr | 96 hr | ||||||
0 | AIMer Concentration (ng/ml) | 1000 | **** | %AIMer Remaining | 100 | **** | |
10-710-610-510-410-3 | 10-210-1100 101 | 800 | 80 | ** | |||
AIMer Concentration (µM) | 600 | *** | 60 | ||||
400 | 40 | ||||||
Stereorandom PS | Stereopure PS | 200 | 20 | ||||
Stereorandom PN | Stereopure PN | ||||||
0 | 0 | ||||||
Stereorandom PS Stereorandom PN | Stereopure PS Stereopure PN |
B | CNS Editing in vivo 1-weekPost-dosing | ||||||||||||||
Human ADAR1-p110Mice | Nonhuman Primates (NHPs) | ||||||||||||||
80 | PBS | 80 | aCSF | ||||||||||||
AIMer | AIMer | ||||||||||||||
Editing | 60 | Editing | 60 | ||||||||||||
40 | 40 | ||||||||||||||
Ugp2 | Ugp2 | ||||||||||||||
B | iPSC-derived Cortical Neurons | Primary Cortical Neurons | ||||
Human Patient-derived | Mecp2R168X KI mouse | |||||
30 | PBS or NTC AIMers | AIMer 1 | 100 | |||
NTC AIMer | ||||||
MECP2 AIMers | Mecp2 AIMer | |||||
Editing | Editing | 80 | ||||
20 | 60 | |||||
% MECP2 | 10 | % Mecp2 | 40 | |||
20 |
convert the Rett Syndrome mutation MECP2R168X into the missense |
R168W |
Editing | 75 |
Editing | 75 | |
B | Cell-free System | Stereopure PS | Ugp2 Editing | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
% | 20 | % | 20 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
0 | 0 | |||||||||||
0 | 20 | 40 | 60 | 80 | 100 | 0.00001 | 0.0001 | 0.001 | 0.01 | 0.1 | 1 | 10 |
codon MECP2 | in human and mouse neuronal cell models. |
• A-to-I(G) editing of RNA-encoding Mecp2R168X restores expression of | |
full-length Mecp2R168W protein in neuronal cells, which correctly | |
colocalizes with heterochromatin. MECP2R168W protein also associates | |
with wild-type MECP2 binding partners, suggesting functionality. |
mRNA | 50 |
%Ugp2 | 25 |
0 |
D
mRNA | 50 | |||
%Ugp2 | 25 | |||
0 | ||||
C | N3U | AIMer-S | AIMer-D | |
Nearest Neighbors |
100 | Stereopure PN | Primary Mouse Hepatocytes | ||||||||||
**** | **** | **** | ||||||||||
**** | ||||||||||||
60 | **** | **** | ||||||||||
80 | **** | **** | ||||||||||
Editing | 40 | * | ** | |||||||||
60 | ||||||||||||
Editing | % | 20 | ||||||||||
0 | ||||||||||||
% | ||||||||||||
40 | Hours | 6 | 24 48 72 96 | 6 | 24 48 72 96 | 6 | 24 48 72 96 | 6 | 24 48 72 96 | |||
0 | 0 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
tex | us | Striatum | m | m | rd | tex | s | Callosum | m | m | Cord | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
u | u | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
r | p | e | llu | o | r | p | e | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
t | lC | t | ll | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
o | m | S | e | o | m | S | e | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
C | oca | ain | b | a | C | oca | in | b | Spinal | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
e | e | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
r | n | a | r | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
pp | r | e | p | pp | Br | e | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
B | C | S | Corpus | C | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Hi | Hi | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
C | Durable CNS Editing in vivo | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Human ADAR1-p110 Mice | PBS | AIMer | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Peak | 30% | >40% | 25% | >40% | 50% | >65% | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
editing | Cortex | Hippocampus | Striatum | Brain Stem | Cerebellum | Spinal Cord | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Ranked by Potency from NTC AIMer
C | Mecp2R168X | WT | ||||
Ladder | AIMer | 1 | PBS | PBS | ||
Mouse | 90 | Mecp2 | ||||
70 | ||||||
Cortical | HDAC2 | |||||
Neurons | 50 | PBS | ||||
Primary Cortical Neurons | ||||||
Dose (µM)
Primary Cortical Neurons
Mecp2R168X KI mouse
DAPI | TUJ1 | Mecp2 |
INTRODUCTION
• PRISM™ generates stereopure oligonucleotides with controlled sequence, |
5′ A
Orphan 3′ A
Base:
C
5′ A 5′ A 5′ A | 5′ C | 5′ C | 5′ G | 5′ G | 5′ G | 5′ U | 5′ U | 5′ U |
3′ C 3′ G 3′ U | 3′ C | 3′ G | 3′ A | 3′ G | 3′ U | 3′ A | 3′ C | 3′ U |
AIMer-S
AIMer-S | AIMer-D |
AIMer Abundance | |
20 | Stereopure PS Primary Mouse Hepatocytes |
Stereopure PN |
6 hr | 96 hr |
80
60
80
60
80
60
80
60
*
*
80
60
*
*
* **
80
60
* |
* |
* |
* |
* | * |
* | |
* |
*
Mecp2R168X KI mouse | ||
DAPI | Mecp2 | Merge |
chemistry, and stereochemistry (Figure 1A).1 |
• PRISM™ can be applied to optimize AIMer design for editing efficiency, |
target sequence, and target tissue. |
Figure 1. Introduction to PRISM™, PN chemistry, and AIMers
N3U
C
N3U
AIMer-D
0 | 10-310-210-1100 | 101 | 1000 | **** | %AIMer Remaining | 80 | *** | ns | |
10-710-610-510-4 | 800 | ||||||||
AIMer Concentration (µM) | AIMer Concentration (ng/ml) | 60 | |||||||
600 | **** | 40 | |||||||
400 | |||||||||
Stereopure PS | Stereopure PN | ||||||||
200 | 20 | ||||||||
AIMer-S | AIMer-S | ||||||||
0 | 0 | ||||||||
AIMer-D | AIMer-D | ||||||||
AIMer-S | AIMer-D | AIMer-S | AIMer-D |
% Ugp2 Editing
40
20
*
40
*
*
*
*
20
* | * |
* | |
* |
*
*
* *
*
*
40
20
* | * | * |
* | ||
* | ||
* | * | |
* |
40
20
* | * | |
* | ||
* | ||
* * | ||
* | 40 | |
* | ||
20 |
* *
*
* *
*
*
*
40
20
**
PBS | NTC |
AIMer 1 | AIMer 1 |
25 | 75 | 25 | 75 | 25 | 75 | 25 | 75 | 25 | 75 | 25 | 75 | 25 | 75 | 25 | 75 | 25 | 75 | 25 | 75 | 25 | 75 | 25 | 75 |
%Ugp2 mRNA Editing
(A, B) Left: Cell-free editing assays: Lysates from 293T cells transfected with human ADAR-p110 (48h) were incubated with either UGP2-targeting AIMer (A) or ACTB-targeting
0
0
0
0
0 |
0
A | O | O B | O | O B | O | O | B | |||||||||
5′ | B | (Rp) | ||||||||||||||
O | O | R | O | O | R | N O | O | R | ||||||||
3′ | 2′ | O | P O | O | B | S P | O | O | B | N P | O | O | B | |||
X | R | N | ||||||||||||||
5′ | B | B Base | O | R | O | R | O | R | ||||||||
R | 2'-Ribose | PO | PS | PN | ||||||||||||
3′ | R | 2′ | X | Stereochemistry and | ||||||||||||
backbone modification | Phosphodiester | Phosphorothioate | Phosphoryl Guanidine | |||||||||||||
B | ADAR Editing of RNA |
A | AIMer | I(G) | |
ADAR | Edited | ||
RNA | RNA | ||
Oligonucleotides can direct A-to-I(G) | ADAR1 is ubiquitously | Cellular reservoir of ADAR | |
RNA editing by recruiting | expressed across tissues, | capacity supports directed editing | |
endogenous ADAR enzymes3 | including liver and CNS | in addition to homeostatic function |
(A) Schematic of approach to improving editing efficiency through AIMer backbone, sugar, and base chemistry. (B, C, D) Primary mouse hepatocytes from human ADAR1-p110 hemizygous mice were treated with 3 μM AIMers (unconjugated), directed toward the Ugp2 mRNA, with variable edit region sequence, chemistry pattern (AIMer-S or AIMer-D), and orphan base (C or N3U) for 72 hours. Ugp2 RNA editing was quantified by Sanger sequencing. (B) Lines connect complexes (represented by circles) with identical 5′- and 3′- nearest neighbors and chemistry format. (C) Lines connect complexes (represented by circles) with identical 5′- and 3′-nearest neighbors and orphan base. Stats: mean of n=3; error bars represent SEM.
- AIMers with orphan site N3U supported higher mean percent RNA editing than AIMers with orphan site C for all nearest neighbor combinations tested, although the magnitude of increase varies (Figure 2B).
- The AIMer-D pattern conferred a higher mean percent RNA editing compared to the AIMer-S patternfor most sequences tested (Figure 2C).
- The impacts of orphan site N3U base modification and the AIMer-D pattern appear largely additive (Figure 2D).
- AIMers with orphan site N3U and the AIMer-D pattern support highly efficient editing for many nearest neighbor combinations in primary mouse hepatocytes.
AIMer (B) at the concentration indicated for 1h, then RNA was extracted from lysates and RNA editing was quantified by Sanger sequencing. Stats: n=3 per dose, per condition; mean ± SEM shown. Right: Primary murine hepatocytes were treated gymnotically with 3 μM Ugp2-targeting AIMers for 6 hours. Cells were refreshed with maintenance media and collected at the indicated time point. RNA editing was quantified by Sanger sequencing. AIMer concentration was quantified by hybridization ELISA 6 hr or 96 hr after the start of the pulse. Stats: A two-way ANOVA was used to calculate statistical significance; * p<0.05,
- p<0.01, *** p<0.001, **** p<0.0001, ns significant.
- Incorporating stereopure PN linkages in AIMers enhances maximum editing compared to either stereopure PS or stereorandom PN in both cell-free and hepatocyte RNA editing assays (Figure 3A).
- The AIMer-D pattern further enhances the editing efficiency benefits of incorporating stereopure PN linkages in AIMers (Figure 3B).
- The AIMer-D pattern does not appear to enhance editing in cell-free systems but does lead to an increased cellular concentration of AIMers immediately after treatment (Figure 3B).
- Collectively, incorporation of stereopure PN linkages and the AIMer-D pattern improve AIMer-mediated RNA editing efficiency. This impact may occur through multiple mechanisms, including enhancing enzyme activity and AIMer uptake.
PBS | 1 | 4 | 8 1216 | PBS | 1 | 4 | 8 1216 | PBS | 1 | 4 | 8 1216 | PBS | 1 | 4 | 8 1216 | PBS | 1 | 4 | 8 1216 | PBS | 1 | 4 | 8 1216 |
Weeks | |||||||||||||||||||||||
ACTB and UGP2 percent editing measured by Sanger sequencing. (A) iNeurons and iAstrocytes were treated gymnotically with ACTB or UGP2 AIMers for 5 days. (B) Left: human ADAR1-p110 mice were administered phosphate buffered saline (PBS) or 100 μg AIMer by intracerebroventricular (ICV) injection (n=5) on day 0 and necropsied on day 7. Right: Cynomolgus monkeys (NHPs) were administered 10 mg ACTB AIMer or artificial CSF (aCSF) by intrathecal administration (n=2) on day 0 and necropsied on day 7. (C) human ADAR1-p110 mice were administered 100 μg AIMer or PBS by ICV injection on day 0 and evaluated for Ugp2 editing across CNS tissues at 1, 4, 8, 12 and 16- weeks post dose. Stats: 2-way ANOVA with post-hoc comparison to PBS (n=5 per time point, per treatment) *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001
- AIMers with the AIMer-S pattern, optimized for the CNS, support dose- dependent editing of ubiquitous housekeeping transcripts in multiple CNS cell lines in vitro (Figure 4A).
- In a human ADAR1-p110 transgenic mouse model, and in NHPs, AIMer- directed editing of housekeeping transcripts were observed across the CNS at one week post single dose (Figure 4B).
- In human ADAR1-p110 mice, AIMer-directed editing peaked at 4 weeks
and persisted 4 months post-single ICV injection (Figure 4C).
(A) Left: Graph adapted from (4) with updated data from RettBASE (data pulled 7 Aug 23). Middle: Proposed MECP2 restoration strategy via
AIMer-basedA-to-I RNA editing. Right: 293T cells transfected (72 h) with FLAG-MECP2WT or FLAG-MECP2R168W, immunoprecipitated with anti-FLAG magnetic beads. Western blot of immunoprecipitated eluates probed for FLAG and NCoR1/SMRT complex members. (B) MECP2 AIMers direct editing in neuronal cells. Percentage editing determined by next-generation sequencing (NGS). Left: Patient iPSC-derived cortical neurons (MECP2R168X) treated gymnotically with 10 μM AIMer (mean ± SEM; n=2 per AIMer). Right: Primary cortical neurons from Mecp2R168X knock-in (KI) mice (E18), treated gymnotically with AIMer for 5 days (mean ± SEM; n=3 per dose/condition). (C) AIMer-based editing of Mecp2R168X restores protein expression. Western blot of nuclear extracts from mouse primary cortical neurons (E18) treated gymnotically with 10 μM AIMer. Primary cortical neurons from Mecp2R168X KI mice (E18) treated with PBS or gymnotic AIMer (30 μM, left; or 1 μM, right) for 5 days. Immunofluorescence staining for nuclei (DAPI, blue) and Mecp2 (magenta) or neuronal marker Tuj1 (Green). Magnification 40X (left) or 10X (right). NTC, nontargeting control. HDAC2, Histone deacetylase 2. WT, wild type.
- We hypothesized that AIMers could be used to correct MECP2R168X, the most common nonsense mutation found in Rett Syndrome (RTT), by converting the premature stop codon to a Tryptophan (W) codon in MECP2 mRNA (Figure 5A).
- We show that exogenous, plasmid-expressed MECP2R168W protein associates with endogenous co-regulatory proteins NCoR1, TBLR1, and HDAC3, suggesting edited MECP2 may retain wild type MECP2 functionality (Figure 5A).
- MECP2 AIMer incorporating the AIMer-D format directs editing of MECP2R168X in human patient-derived cortical neurons and primary cortical neurons isolated from the Mecp2R168X KI mouse (Figure 5B).
- MECP2 AIMer incorporating the AIMer-D pattern restores Mecp2 protein expression and localization in primary cortical neurons isolated from the Mecp2R168X KI mouse (Figure 5C).
References: 1. Kandasamy, et al., 2022. Nuc Acids Res 50(10):5443-5466; 2. Monian et al., 2022 Nature Biotech 40(7):1093-1102. doi: 10.1038/s41587-022-01225-1; 3. Woolf, et al., 1995 Proc Natl Assoc Sci 92:8298-8302; 4. Krishnaraj, et al., Hum Mutat 2017;00:1-10.Acknowledgments: The authors are grateful to Nicole Neuman (Wave Life Sciences) and Eric Smith for editorial and graphical support, respectively. This work was funded by Wave Life Sciences. Patient fibroblast cells were kindly provided by Rett Syndrome Research Trust and Harvard Stem Cell Institute iPS Core Facility.
Presented at the 27th Annual Meeting of the American Society of Gene & Cell Therapy, May 7-11, 2024 - Baltimore, MD | Supported by Wave Life Sciences, Cambridge, MA, USA |
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