RNA-driven Evolution of AAV Capsid Libraries Identifies ...
Transcript of RNA-driven Evolution of AAV Capsid Libraries Identifies ...
© 2021 Voyager Therapeutics, Inc. – Not for Distribution.1
RNA-driven Evolution of AAV Capsid
Libraries Identifies Variants with High
Transduction Efficiency in Non-Human
Primate Central Nervous SystemMathieu Nonnenmacher, PhD
ASGCT 2021
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
Disclosure
• Mathieu Nonnenmacher is a full-time employee of Voyager Therapeutics,
Cambridge, MA
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ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.3
TRACER
Library
AAAAA
Library
*Library mRNA
AAAA
AAAA
AAAA
NGS +
Subcloning
Library RNA (brain)
AAV LibraryCapsid DNA Library
©
Neu CAP
TRACER-NHP Directed Evolution for Neurotrophic AAV Capsids
IV Dosing
• Challenges with widespread CNS gene delivery
• Current best-in-class, AAV9, achieves only limited
CNS transduction from IV route
• Local delivery can unlock some indications, but may
not achieve sufficient distribution for others
• Functional evolution of AAV in mice identified BBB-
penetrant AAV capsids but failed to translate to
primates (Deverman, 2016; Hordeaux et al., 2018)
• TRACER platform validated in rodents
(Nonnenmacher et al., 2020) for functional
biopanning
• Voyager strategy: apply TRACER platform for
RNA-driven AAV9 evolution in primate brainAdapted from
Nonnenmacher et al., Mol Ther Methods Clin Dev. 2020
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
Cross-Species TRACER Evolution
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Stepwise NGS analysis
CNS mRNA
Enrichment
Analysis
NHP Brain P1 NHP Brain P2
Virus Library
Human Brain Microvascular Endothelial Cells
Mouse Brain P1 Mouse Brain P2 Mouse Brain P3
HSC7
HSC17
PHP.B
AAV9
PHP.eB
NHP
(1800)
hBMVEC
(100)
Mouse
(100)
SYNTHETIC
POOL
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
Validation of Top TRACER Candidates in NHP
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Pooled Brain RNA Enrichment Screen
Adult cyno Macaques (n=2)
• Intravenous dosing
• 2e13 vg/kg (low dose)
• 14 days in-life
• Transgene Expression
Individual Validation (5 candidates)
ssAAV CBA-TG-HAtag
9P8019P802
9P804
9P805
9P803*
*Selected from
alternate screen
HSC17 (2.53x)
PHP.B (1.23x)
AAV9 (1x)
1
52
>10x
PHP.eB (2.52x)
Fold over AAV9 Fold over AAV9
NHP1 NHP2
1 2 3 1 2 3
NHP1 NHP2
1 2 3 1 2 3
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
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0.0001
0.001
0.01
0.1
1
10
100m
RN
A (
fold
vs T
BP
)
TRACER-NHP Capsids Show Increased Transgene mRNA Expression in the Brain
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1,000-10,000x
2-6x
5-40x
7-30x
2-7x
wtAAV9
9P801
9P802
9P803
9P804
9P805
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
SC-c
ervica
l
SC-T
hora
cic
SC-L
umba
r
SC-c
ervica
l
SC-T
hora
cic
SC-L
umba
r0.001
0.01
0.1
1
10
100
mR
NA
(fo
ld v
s T
BP
)
Capsid 9P801 Mediates Strong Transduction in the Spinal Cord
9P801 Spinal Cord Dorsal horn
Ventral horn
L2
T6
C2
Clarke’s column
Dorsal horn
Ventral horn
Clarke’s column
AAV9 Spinal Cord
L2
T6
C2100-1000x
wtAAV9
9P801
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
Transduction by AAV9 and 9P801 in the DRG
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Lumbar L2
Thoracic T12
9P801AAV9
Cervical C2
10x
DRG-c
ervica
l
DRG-T
hora
cic
DRG-L
umba
r
DRG-c
ervica
l
DRG-T
hora
cic
DRG-L
umba
r0.1
1
10
100
mR
NA
(fo
ld v
s T
BP
)
wtAAV9
9P801
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
9P801 Mediates Widespread Transgene Expression in NHP Brain
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9P801
AAV9
Slice # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Slice #7 Slice #8 Slice #12
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
9P801 Displays a Predominantly Neuronal Tropism in Deep Cerebellar Nuclei
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9P801– NHP#1 9P801– NHP#2
9P801 – NHP#1 9P801 – NHP#2
AAV9– NHP#1
AAV9– NHP#1 AAV9– NHP#1
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
9P801 Displays a Predominantly Neuronal Tropism in the Hippocampus
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9P801– NHP#1 9P801– NHP#2
9P801 – NHP#1 9P801 – NHP#2
AAV9– NHP#1
AAV9– NHP#1 AAV9– NHP#1
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
9P801 Displays a Predominantly Neuronal Tropism in the Thalamus (Lateral Geniculate Nucleus)
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9P801– NHP#1 9P801– NHP#2
9P801 – NHP#1 9P801 – NHP#2
AAV9– NHP#1
AAV9– NHP#1 AAV9– NHP#1
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
Capsid 9P801 Displays Strong Transduction in Multiple CNS Regions
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Putamen Globus Pallidus Somatosensory cortex
Hippocampus
Caudate ThalamusPontine grey matter Dentate nucleus
Lateral Reticular Nucleus Cuneate nucleus Cerebellar cortex
Temporal cortex
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
Transduction of Peripheral Tissues by 9P801 and 9P804
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Liver
Quadriceps
Heart
9P801 9P804AAV9
DRG
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
Key Takeaways
• The first run of TRACER platform in NHPs identified several BBB-penetrating capsids
• The 9P801 capsid allows unprecedented levels of primate CNS transduction by IV
dosing, no toxicity was observed in the liver, spinal cord or DRG
• The 9P801 capsid showed no major difference with AAV9 in non-CNS tissue
transduction, the 9P804 capsid displayed strong heart transduction and significant
DRG detargeting, other candidates still under evaluation
• Nine additional NHP libraries are currently in late screening or characterization phase
•
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The TRACER-NHP platform holds strong potential for development of enhanced
capsids across multiple organisms, attributes and target tissues
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
Thank you
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Amy TylerXiao-Qin
Jing
ShaoyongXiaodong
MattJiangyu
Katie
Wei
ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.
Thank you
• Nilesh Pande (IHC)
• June Deng
• Jianyu Chang
• Michael Hefferan
• Omar Khwaja
• Steve Paul
• Jay Hou
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Contact and links:
https://www.voyagertherapeutics.com/
https://www.voyagertherapeutics.com/join-our-team/careers/