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

2

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


Cross-Species TRACER Evolution

4

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


Validation of Top TRACER Candidates in NHP

5

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


Front

al C

orte

x

Sen

sory

Cor

tex

Mot

or C

orte

x

Put

amen

Thalam

us

Cer

ebellar C

orte

x

Front

al C

orte

x

Sen

sory

Cor

tex

Mot

or C

orte

x

Put

amen

Thalam

us

Cer

ebellar C

orte

x

Front

al C

orte

x

Sen

sory

Cor

tex

Mot

or C

orte

x

Put

amen

Thalam

us

Cer

ebellar C

orte

x

Front

al C

orte

x

Sen

sory

Cor

tex

Mot

or C

orte

x

Put

amen

Thalam

us

Cer

ebella

r Cor

tex

Front

al C

orte

x

Sen

sory

Cor

tex

Mot

or C

orte

x

Put

amen

Thalam

us

Cer

ebellar C

orte

x

Front

al C

orte

x

Sen

sory

Cor

tex

Mot

or C

orte

x

Put

amen

Thalam

us

Cer

ebellar C

orte

x

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

6

1,000-10,000x

2-6x

5-40x

7-30x

2-7x

wtAAV9

9P801

9P802

9P803

9P804

9P805


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


Transduction by AAV9 and 9P801 in the DRG

8

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


9P801 Mediates Widespread Transgene Expression in NHP Brain

9

9P801

AAV9

Slice # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Slice #7 Slice #8 Slice #12


9P801 Displays a Predominantly Neuronal Tropism in Deep Cerebellar Nuclei

10

9P801– NHP#1 9P801– NHP#2

9P801 – NHP#1 9P801 – NHP#2

AAV9– NHP#1

AAV9– NHP#1 AAV9– NHP#1


9P801 Displays a Predominantly Neuronal Tropism in the Hippocampus

11

9P801– NHP#1 9P801– NHP#2

9P801 – NHP#1 9P801 – NHP#2

AAV9– NHP#1



9P801 Displays a Predominantly Neuronal Tropism in the Thalamus (Lateral Geniculate Nucleus)

12

9P801– NHP#1 9P801– NHP#2

9P801 – NHP#1 9P801 – NHP#2

AAV9– NHP#1



Capsid 9P801 Displays Strong Transduction in Multiple CNS Regions

13

Putamen Globus Pallidus Somatosensory cortex

Hippocampus

Caudate ThalamusPontine grey matter Dentate nucleus

Lateral Reticular Nucleus Cuneate nucleus Cerebellar cortex

Temporal cortex


Transduction of Peripheral Tissues by 9P801 and 9P804

14

Liver

Quadriceps

Heart

9P801 9P804AAV9

DRG


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

•

15

The TRACER-NHP platform holds strong potential for development of enhanced

capsids across multiple organisms, attributes and target tissues


Thank you

16

| 16 |

Amy TylerXiao-Qin

Jing

ShaoyongXiaodong

MattJiangyu

Katie

Wei


Thank you

• Nilesh Pande (IHC)

• June Deng

• Jianyu Chang

• Michael Hefferan

• Omar Khwaja

• Steve Paul

• Jay Hou

17

Contact and links:

[email protected]

https://www.voyagertherapeutics.com/

https://www.voyagertherapeutics.com/join-our-team/careers/

mailto:[email protected]

https://www.voyagertherapeutics.com/

https://www.voyagertherapeutics.com/join-our-team/careers/

rna-driven evolution of aav capsid libraries identifies

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